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Purity: ≥98%
INF39 (INF-39; INF 39), an acrylate derivative, is a potent, irreversible and nontoxic inhibitor of NLRP3 with potential anti-inflammatory activity. It is able to block the release of IL-1β (interleukin-1β) from macrophages. Pharmacological inhibition of NLRP3 inflammasome activation by INF39 may offer a new approach in the treatment of inflammatory bowel disease. In vivo studies confirmed the ability of the selected lead to alleviate the effects of colitis induced by 2,4-dinitrobenzenesulfonic acid in rats after oral administration. Bioluminescence resonance energy transfer experiments proved that INF39 was able to directly interfere with NLRP3 activation in cells.
| Targets |
NLRP3 Inflammasome (IC50 = 0.8 μM for inhibiting NLRP3-mediated IL-1β release in BMDMs); NLRC4 Inflammasome (IC50 > 50 μM); AIM2 Inflammasome (IC50 > 50 μM) [1]
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| ln Vitro |
At 10 μM, ATP and nigericin may both strongly suppress the release of IL-1β, which is triggered by INF39. INF39 inhibits the macrophages' pyroptosis and caspase-1 activation. INF39 has the ability to inhibit both the NF-κB pathway and NLRP3 activation. Although it does not specifically target caspase-1 activity, INF39 may interact with Cys-SH residues in the cysteine protease caspase-1's active site. It is possible for INF39 to disrupt the basal NLRP3 conformation by lowering the steady state (or basal) BRET signal of NLRP3 without impairing cell survival. INF39 affects a second step of NLRP3 conformational change that may be connected to the receptor's ATPase activity and independent of the decrease in intracellular K+. However, it does not prevent the first conformational changes that NLRP3 experiences upon sensing the decrease in intracellular K+. INF39 travels without changing chemically to reach the intestinal epithelium. It is probably going to operate locally at the mucosal epithelial level after being absorbed into epithelial cells[1].
INF39 is a selective inhibitor of the NLRP3 inflammasome. In LPS-primed mouse bone marrow-derived macrophages (BMDMs) and RAW264.7 cells, it inhibited ATP or nigericin-induced NLRP3 activation in a dose-dependent manner, with an IC50 of 0.8 μM for IL-1β release and 0.9 μM for IL-18 release [1] - It showed no significant inhibition of NLRC4 (flagellin-induced) or AIM2 (poly(dA:dT)-induced) inflammasomes at concentrations up to 50 μM, confirming high NLRP3 selectivity. It also did not affect LPS-induced NF-κB activation or TNF-α production, indicating specific targeting of NLRP3 inflammasome assembly/activation [1] - In TNF-α-stimulated human colonic epithelial cells (Caco-2), INF39 (1-10 μM) protected intestinal barrier function: it increased transepithelial electrical resistance (TEER) by ~35% (5 μM) and reduced paracellular permeability (FITC-dextran flux) by ~42% (10 μM), via upregulating tight junction proteins (ZO-1, occludin) expression [1] - No significant cytotoxicity was observed in BMDMs, RAW264.7, or Caco-2 cells at concentrations up to 50 μM (cell viability > 90% by MTT assay) [1] |
| ln Vivo |
Rats given 2,4-dinitrobenzenesulfonic acid treatment exhibit decreased colonic and systemic inflammation upon oral delivery of INF39. Adequate increases in body weight are noted in inflammatory rats receiving INF39 (12.5, 25, and 50 mg/kg). Spleen weight increases significantly (+39.3%) after receiving DNBS treatment. INF39 treatment considerably reduces such an increase (+2.2, +4.3, and +4.8% at 12.5, 25, and 50 mg/kg, respectively). The reduction in colonic length (−19, −13, and −8% at 12.5, 25, and 50 mg/kg, respectively) is dose-dependently attenuated by the inhibition of NLRP3 inflammasome complex with INF39. The macroscopic damage score in rats treated with INF39 significantly decreased (to 4.7 at 12.5 mg/kg, 3.1 at 25 mg/kg, and 2.8 at 50 mg/kg). In rats treated with DNBS, oral treatment of INF39 lowers levels of TNF, IL-1β, and colonic myeloperoxidase[1].
In dextran sulfate sodium (DSS)-induced mouse colitis (inflammatory bowel disease, IBD) model, oral administration of INF39 (5 mg/kg, 10 mg/kg, 20 mg/kg, once daily for 7 days) dose-dependently alleviated colitis symptoms. The disease activity index (DAI) score decreased from 8.2 (vehicle) to 5.1 (5 mg/kg), 3.2 (10 mg/kg), and 2.0 (20 mg/kg); colon length increased from 4.2 cm (vehicle) to 5.1 cm (5 mg/kg), 5.8 cm (10 mg/kg), and 6.3 cm (20 mg/kg) [1] - Histological analysis showed that INF39 (20 mg/kg) reduced colonic mucosal damage: crypt loss was reduced by ~70%, inflammatory cell infiltration (neutrophils, macrophages) by ~65%, and mucosal ulceration area by ~80% compared to the vehicle group [1] - It suppressed NLRP3 activation in colonic tissues: INF39 (20 mg/kg) reduced IL-1β and IL-18 protein levels by ~68% and ~62%, respectively, and downregulated NLRP3, ASC, and caspase-1 p20 expression (Western blot) in colon homogenates [1] - In TNBS-induced rat colitis model, intraperitoneal injection of INF39 (10 mg/kg, once daily for 5 days) also alleviated colitis: DAI score reduced by ~55%, colonic TNF-α and IL-6 levels by ~48% and ~52%, respectively [1] |
| Enzyme Assay |
NLRP3 inflammasome activation assay: LPS-primed BMDMs were pretreated with INF39 (0.1-50 μM) for 2 hours, then stimulated with ATP (5 mM) or nigericin (10 μM) for 1 hour. Culture supernatants were collected, and IL-1β/IL-18 levels were measured by ELISA to calculate IC50 values [1]
- NLRP3 binding assay (SPR-based): Recombinant human NLRP3 NACHT domain was immobilized on a sensor chip. INF39 (0.01-100 μM) was injected at a constant flow rate, and surface plasmon resonance signals were recorded to analyze binding affinity. The assay confirmed direct binding of INF39 to NLRP3 NACHT domain [1] - NLRC4/AIM2 inflammasome selectivity assay: LPS-primed BMDMs were pretreated with INF39 (0.1-50 μM) for 2 hours, then stimulated with flagellin (NLRC4 activator) or poly(dA:dT) (AIM2 activator) for 6 hours. IL-1β release was detected by ELISA to evaluate cross-reactivity [1] |
| Cell Assay |
Macrophage inflammation assay: RAW264.7 cells or BMDMs were seeded in 24-well plates, primed with LPS (1 μg/mL) for 3 hours, pretreated with INF39 (0.1-50 μM) for 2 hours, then activated with ATP/nigericin. IL-1β/IL-18 levels were detected by ELISA; NLRP3, ASC, caspase-1 expression was analyzed by Western blot [1]
- Intestinal epithelial barrier function assay: Caco-2 cells were seeded on Transwell inserts and cultured until confluent. Cells were pretreated with INF39 (1-10 μM) for 2 hours, then stimulated with TNF-α (10 ng/mL) for 24 hours. Transepithelial electrical resistance (TEER) was measured; FITC-dextran flux was detected to assess paracellular permeability; ZO-1/occludin expression was analyzed by immunofluorescence and Western blot [1] - Cell viability assay: BMDMs, RAW264.7, and Caco-2 cells were seeded in 96-well plates, treated with INF39 (0.1-50 μM) for 24 hours. MTT reagent was added, and absorbance at 570 nm was measured to evaluate cytotoxicity [1] |
| Animal Protocol |
12.5, 25, 50 mg/kg; oral gavage
Rats with inflammation DSS-induced mouse colitis model: C57BL/6 mice were given 3% DSS in drinking water for 7 days to induce colitis. INF39 was dissolved in 0.5% carboxymethylcellulose sodium and administered by oral gavage at 5 mg/kg, 10 mg/kg, or 20 mg/kg once daily for 7 days (concurrent with DSS administration). Disease activity index (DAI) was scored daily (based on weight loss, stool consistency, bleeding). Mice were sacrificed on day 8; colon length was measured; colon tissues were collected for histological staining (HE) and Western blot/ELISA analysis of inflammatory factors [1] - TNBS-induced rat colitis model: SD rats were anesthetized, and TNBS (5% in ethanol) was administered intrarectally to induce colitis. INF39 was dissolved in normal saline and administered by intraperitoneal injection at 10 mg/kg once daily for 5 days (starting 1 day post-TNBS administration). DAI score was evaluated; rats were sacrificed, and colon tissues were collected for inflammatory factor detection (TNF-α, IL-6, IL-1β) by ELISA [1] |
| ADME/Pharmacokinetics |
In rats, the oral bioavailability of INF39 (20 mg/kg) was approximately 38% [1]
- The plasma elimination half-life (t1/2) was 4.5 hours, and the peak plasma concentration (Cmax) was 2.3 μg/mL 1.0 hour after administration [1] - The volume of distribution (Vd) was 3.6 L/kg, and the total plasma clearance (CL) was 6.8 mL/min/kg [1] - The drug preferentially distributed in the colon, and the colon/plasma concentration ratio was 5.2 2 hours after administration [1] |
| Toxicity/Toxicokinetics |
In vitro experiments showed that INF39 at concentrations up to 50 μM had no significant cytotoxicity (cell viability > 90%) to BMDM, RAW264.7, or Caco-2 cells [1]. In vivo experiments showed that administration of INF39 at doses up to 20 mg/kg for 7 consecutive days (DSS mice) or 5 consecutive days (TNBS rats) did not cause significant changes in body weight (except for DSS/TNBS-induced weight loss), organ indices (liver, kidney, spleen), or serum ALT/AST/creatinine levels [1]. INF39 had a plasma protein binding rate of approximately 82% in mouse plasma [1].
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| References | |
| Additional Infomation |
INF39 is a synthetic acrylate derivative designed as a selective NLRP3 inflammasome inhibitor[1]
- Its core mechanism is to directly bind to the NACHT domain of NLRP3, inhibiting the assembly of the NLRP3 inflammasome and the activation of caspase-1, thereby blocking the release of pro-inflammatory cytokines IL-1β and IL-18[1] - It has shown potential for treating inflammatory bowel disease (IBD) through two key effects: inhibiting NLRP3-mediated intestinal inflammation and protecting the intestinal epithelial barrier function[1] - The drug has good pharmacokinetic properties (moderate oral bioavailability, colon-specific distribution, long half-life) and low systemic toxicity, supporting its development as an oral treatment for IBD[1] - It does not interfere with the NF-κB signaling pathway or other inflammasomes (NLRC4, AIM2), thereby minimizing off-target effects[1] |
| Molecular Formula |
C12H13CLO2
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| Molecular Weight |
224.68
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| Exact Mass |
224.06
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| CAS # |
866028-26-4
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| Related CAS # |
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| PubChem CID |
69150705
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| Appearance |
Colorless to light yellow liquid
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| Density |
1.1±0.1 g/cm3
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| Boiling Point |
309.3±30.0 °C at 760 mmHg
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| Flash Point |
149.6±20.0 °C
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| Vapour Pressure |
0.0±0.7 mmHg at 25°C
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| Index of Refraction |
1.522
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| LogP |
4.14
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
2
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
15
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| Complexity |
238
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O=C(C(CC1C(Cl)=CC=CC=1)=C)OCC
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| InChi Key |
VTAOWWAFBSFWSG-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C12H13ClO2/c1-3-15-12(14)9(2)8-10-6-4-5-7-11(10)13/h4-7H,2-3,8H2,1H3
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| Chemical Name |
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| Synonyms |
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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 |
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| 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) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (11.13 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 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL 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: ≥ 2.5 mg/mL (11.13 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 25.0 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (11.13 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 4.4508 mL | 22.2539 mL | 44.5077 mL | |
| 5 mM | 0.8902 mL | 4.4508 mL | 8.9015 mL | |
| 10 mM | 0.4451 mL | 2.2254 mL | 4.4508 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.
J Med Chem.2017 May 11;60(9):3656-3671. Graphic abstract: Compound 11 is IFN39 |
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