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| Targets |
The target is the NLRP3 (NOD-like receptor family, pyrin domain containing 3) protein, a key component of the NLRP3 inflammasome complex. NLRP3-IN-10 directly interacts with NLRP3 to suppress LPS-induced NLRP3 priming and activation, preventing the assembly of the inflammasome complex and subsequent activation of caspase-1.
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| ln Vitro |
NLRP3-IN-10 (compound 14c) (0.4, 1.6, 6.4 μM; 40 min), has a dose-dependent, substantial inhibitory effect on the activation of the NLRP3 inflammasome in THP-1 cells caused by LPS-MSU (12 h). THP-1 cells are not cytotoxic when exposed to NLRP3-IN-10 (0.1–6.4 μM; 1.5 hours), and it prevents pyroptosis caused by nigericin (0.1 and 0.4 μM; 40 minutes) [1]. In THP-1 cell supernatants, NLRP3-IN-10 (0.1, 0.2, and 0.4 μM; 40 min) dose-dependently decreases caspase-1 p20 and IL-1β processing [1]. In THP-1 cells, NLRP3-IN-10 (0.2 μM and 0.8 μM; 40 min) decreases the rate of ASC puncta, indicating disruption of ASC oligomerization, and inhibits LPS-induced THF-α (3 μM and 5 μM; 40 min). >[1]. It is believed that the NLRP3 inflammasome functions in two stages: initiation and activity. By directly interacting with NLRP3, NLRP3-IN-10 (1, 10 and 100 μM; 40 min) suppresses the initiation of NLRP3 triggered by LPS [1].
In biochemical assays, NLRP3-IN-10 has an IC50 of 251.1 nM for inhibiting IL-1beta release. In cellular models (THP-1 cells), it dose-dependently (0.2-0.8 microM) reduces the percentage of ASC speck-positive cells. At concentrations of 0.1-0.4 microM, it reduces caspase-1 p20 and IL-1beta processing. At 0.4-6.4 microM over 40 minutes, it has a substantial inhibitory effect on NLRP3 activation induced by LPS/MSU. |
| ln Vivo |
NLRP3-IN-10 (compound 14c) (10 mg/kg; i.v.; single dose) decreased spleen IL and peritoneal neutrophil influx in mice with MSU-induced peritonitis-1β in an LPS-triggered animal model[1]. After oral administration at various doses, NLRP3-IN-10 (10, 30, 90 mg/kg; single dose) demonstrated exceptionally low exposure (14.6−23.53 μg·h/L), poor bioavailability (2.47−13.79%), and high plasma clearance (2201.58 −5551.12 L/h/kg) [1]. NLRP3-IN-10 pharmacokinetics in mice [1] The dosage by route (mg/kg) AUC0-t (μg·h/L) CL in terms of L/h/kg Cmax in μg/L T1/ 2 (hour) Tmax in hours F (%) IV 10. 105.88 133.75 81.97 3.13 0.11 PO 10 14.60 2201.58 3.35 7.43 2.11 13.79 PO 30 15.84 2583.27 16.42 7.92 1.26 4. 99 PO 90 23.53 5551.12 13.59 6.08 4.21 2.
In an MSU-induced peritonitis mouse model, a single intravenous dose of 10 mg/kg of NLRP3-IN-10 reduces peritoneal neutrophil influx and IL-1beta levels in the spleen. This demonstrates that the compound is effective in vivo in a model of acute inflammation, specifically by blocking the NLRP3 pathway. |
| Enzyme Assay |
Biochemical assays for NLRP3 typically use HEK293T cells overexpressing NLRP3 and ASC, which are often used for screening. After compound treatment, inflammasome activation is induced by nigericin. The cell supernatant is collected and IL-1beta release is measured using a human IL-1beta ELISA kit. The IC50 is calculated to quantify the inhibition of inflammasome activity.
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| Cell Assay |
The standard cellular protocol uses THP-1 cells differentiated into macrophages with PMA. Cells are primed with LPS (e.g., 1 microg/mL for 3 hours), then treated with varying concentrations of NLRP3-IN-10 (0.1-6.4 microM) for 40 minutes, followed by activation with ATP (5 mM) or MSU crystals. After incubation, caspase-1 activation in the supernatant is assessed by Western blotting for the p20 subunit. IL-1beta and IL-18 levels in the supernatant are quantified by ELISA. ASC speck formation is visualized by immunofluorescence.
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| Animal Protocol |
Animal/Disease Models: LPS-induced MSU Induced peritonitis mouse model (C57BL/6J mice, 7 weeks old, male) [1] LPS: 1 mg/kg, intraperitoneal (ip) injection; MSU: 100 mg/kg, intravenous (iv) (iv)injection
Doses: 10 mg/kg Route of Administration: intravenous (iv) (iv)injection; single dose Experimental Results: 6 hrs (hrs (hours)) after treatment, the release of IL-1β in the spleen of mice was Dramatically diminished. The increase in peritoneal neutrophil influx was Dramatically diminished compared with controls. In an MSU-induced peritonitis mouse model, BALB/c mice are primed with an intraperitoneal (i.p.) injection of LPS. After priming, MSU crystals are injected i.p. to induce acute peritonitis. NLRP3-IN-10 is administered via intravenous injection at a dose of 10 mg/kg as a single dose. After 6 hours, peritoneal lavage fluid is collected. Neutrophil infiltration is counted by flow cytometry or hemocytometer, and IL-1beta concentration in the lavage fluid and spleen is measured by ELISA. |
| ADME/Pharmacokinetics |
After oral administration in mice at doses of 10, 30, and 90 mg/kg, the compound exhibits very low systemic exposure with an AUC (Area Under the Curve) of only 14.6-23.53 microg·h/L. It shows poor oral bioavailability (2.47-13.79%) and high plasma clearance (2201.58-5551.12 L/h/kg). This low oral bioavailability suggests that intravenous administration is the preferred route for in vivo efficacy studies.
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| Toxicity/Toxicokinetics |
Specific toxicological data for NLRP3-IN-10 are not detailed in the available literature. However, its low systemic exposure after oral administration and its mechanism specifically targeting the NLRP3 inflammasome suggest a potential for localized activity with reduced systemic toxicity in certain disease contexts. Standard histopathological analysis would be performed in vivo.
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| References | |
| Additional Infomation |
NLRP3-IN-10 is a potent research tool for studying NLRP3-related inflammatory diseases. Despite having poor oral bioavailability, its efficacy in an MSU-induced peritonitis model via IV administration validates NLRP3 as a therapeutic target for acute inflammation. As of the latest updates, this compound has not progressed to clinical trials or been approved for sale.
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| Molecular Formula |
C17H14BRFO3
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|---|---|
| Molecular Weight |
365.19
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| Exact Mass |
364.011
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| CAS # |
2641826-39-1
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| PubChem CID |
156702996
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| Appearance |
Light yellow to yellow solid powder
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| LogP |
4.4
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
22
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| Complexity |
393
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C(/C1C=C(OC)C(Br)=CC=1OC)=C\C(C1C=CC(F)=CC=1)=O
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| InChi Key |
ZXMIFRJYIRYWTC-VMPITWQZSA-N
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| InChi Code |
InChI=1S/C17H14BrFO3/c1-21-16-10-14(18)17(22-2)9-12(16)5-8-15(20)11-3-6-13(19)7-4-11/h3-10H,1-2H3/b8-5+
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| Chemical Name |
(E)-3-(4-bromo-2,5-dimethoxyphenyl)-1-(4-fluorophenyl)prop-2-en-1-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 : ~100 mg/mL (~273.83 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 | 2.7383 mL | 13.6915 mL | 27.3830 mL | |
| 5 mM | 0.5477 mL | 2.7383 mL | 5.4766 mL | |
| 10 mM | 0.2738 mL | 1.3692 mL | 2.7383 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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