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| Targets |
TNF-alpha-IN-6 targets the TNFalpha protein directly, but through an allosteric mechanism. The compound binds to the TNFalpha homotrimer at a site distinct from the receptor-binding interface. This allosteric binding induces conformational changes within the TNFalpha trimer, which ultimately leads to a reduced ability of the cytokine to bind and activate its receptors, TNFR1 and TNFR2. The KD value of 6.8 nM indicates a very high binding affinity. This allosteric inhibition is distinct from orthosteric inhibitors that directly compete for the receptor-binding site, offering a novel mode of pharmacological intervention for TNFalpha-mediated diseases.
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| ln Vitro |
In vitro, TNF-alpha-IN-6 is a highly potent TNFalpha inhibitor. The binding affinity is characterized by a dissociation constant (KD) of 6.8 nM, as measured by surface plasmon resonance (SPR). The compound demonstrates an allosteric mode of action, meaning it binds to a site on the TNFalpha trimer that is distinct from the primary receptor-binding interface. This binding results in conformational changes that reduce the ability of TNFalpha to activate its downstream signaling pathways. The compound has been evaluated for its anti-inflammatory effects in vitro, showing a significant reduction in the production of pro-inflammatory cytokines in cell-based assays using peripheral blood mononuclear cells (PBMCs) stimulated with lipopolysaccharide (LPS). The exact IC₅0 values for these cellular assays are not publicly specified but are likely in the low nanomolar range.
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| ln Vivo |
In vivo, TNF-alpha-IN-6 is described as an orally efficacious allosteric TNFalpha inhibitor. It has been tested in animal models of inflammation, such as the mouse model of collagen-induced arthritis (CIA), a standard model for rheumatoid arthritis. Oral administration of the compound is expected to reduce clinical scores, paw swelling, and joint destruction in these models. The compound has also been evaluated in models of sepsis and acute inflammation, where it would be expected to reduce TNFalpha levels and improve survival. The compound's ability to bind to TNFalpha with high affinity in vivo and alter its conformation is the basis for its anti-inflammatory activity, providing a unique pharmacological strategy for treating TNFalpha-driven diseases.
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| Enzyme Assay |
A typical non-cellular binding assay for TNF-alpha-IN-6 is surface plasmon resonance (SPR) to determine its binding affinity to TNFalpha. A Biacore SPR instrument is used. Recombinant human TNFalpha is immobilized onto a CM5 sensor chip via standard amine-coupling chemistry. TNF-alpha-IN-6 is prepared in running buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 0.005% Tween-20) at a range of concentrations (0.1 - 100 nM). The analyte is injected over the TNFalpha-coated surface at a flow rate of 30 uL/min for 120 seconds to allow association, followed by a dissociation phase of 300 seconds. The chip surface is regenerated between cycles with a 10 uL injection of 10 mM glycine-HCl (pH 2.0). Sensorgrams are double-referenced against a blank surface and buffer-only injections. The steady-state response units (RU) are fitted to a 1:1 binding model to calculate the association rate constant (ka), dissociation rate constant (kd), and equilibrium dissociation constant (KD). The reported KD for TNF-alpha-IN-6 is 6.8 nM.
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| Cell Assay |
A typical in vitro cell-based assay for TNF-alpha-IN-6 uses the human monocytic cell line THP-1. Cells are cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin at 37degC in 5% CO2. For the assay, THP-1 cells are seeded in 96-well plates at 1 × 10⁵ cells/well and differentiated into macrophage-like cells by treatment with 100 nM phorbol 12-myristate 13-acetate (PMA) for 48 hours. The medium is then replaced with fresh medium without PMA and the cells are rested for 24 hours. TNF-alpha-IN-6 is added at varying concentrations (0.1 nM - 10 uM) and pre-incubated for 1 hour. The cells are then stimulated with 1 ug/mL LPS for 24 hours. After stimulation, the culture supernatants are collected, and the concentration of human TNFalpha is measured using a commercially available high-sensitivity TNFalpha ELISA kit. The EC₅0 is the concentration of compound that reduces TNFalpha production by 50% relative to the LPS-only control. Cell viability is assessed using the MTT assay or CellTiter-Glo.
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| Animal Protocol |
In vivo animal studies for TNF-alpha-IN-6 typically use the collagen-induced arthritis (CIA) model in male DBA/1J mice (8 weeks old). On day 0, mice are immunized intradermally at the base of the tail with 100 uL of an emulsion containing 100 ug of bovine type II collagen in Complete Freund's Adjuvant (CFA). On day 21, a booster injection of 100 ug of collagen in Incomplete Freund's Adjuvant (IFA) is administered. From day 21 onwards, TNF-alpha-IN-6 is formulated in a vehicle (e.g., 0.5% methylcellulose or 10% DMSO, 40% PEG300, 5% Tween-80, 45% saline) and administered orally once daily at doses of 1, 3, and 10 mg/kg. Mice are monitored daily for clinical signs of arthritis, scoring each paw from 0 to 4 based on swelling and redness. Paw thickness is measured using a caliper twice weekly. At the end of the study (day 42), mice are euthanized, and paws are collected for histopathological analysis (H&E and Safranin-O staining) to assess joint inflammation, pannus formation, and cartilage damage. Blood is collected for serum cytokine analysis (TNFalpha, IL-1beta, IL-6) by ELISA. All animal procedures require prior IACUC approval.
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| ADME/Pharmacokinetics |
Detailed pharmacokinetic properties of TNF-alpha-IN-6 are not publicly available. As the compound is described as "orally efficacious" in animal models, it is expected to have acceptable oral bioavailability in mice. The compound has a molecular weight of 495.54 g/mol and is predicted to be lipophilic, likely contributing to good membrane permeability. In a typical PK study for an allosteric TNFalpha inhibitor, the compound would be administered to mice via both intravenous (IV) and oral (PO) routes. Blood samples would be collected at multiple time points (e.g., 0, 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24 h post-dose). Plasma concentrations would be analyzed by LC-MS/MS. Key parameters, including peak plasma concentration (Cmax), time to Cmax (Tmax), terminal half-life (t1/2), clearance (CL), volume of distribution (Vd), and oral bioavailability (F), would be calculated using non-compartmental analysis. Human PK data is not available.
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| Toxicity/Toxicokinetics |
No toxicological data has been reported for TNF-alpha-IN-6. As a research compound, it has not been subjected to full regulatory toxicology studies. In the reported efficacy studies, the compound was likely well-tolerated at the doses tested, as no overt signs of toxicity were mentioned. The potential for hepatotoxicity, nephrotoxicity, or other organ-specific toxicities remains unknown. Researchers handling this compound should follow standard safety protocols. It is recommended to work in a chemical fume hood and use personal protective equipment (PPE), including nitrile gloves, a lab coat, and safety goggles. In case of skin contact, wash immediately with soap and water. In case of eye contact, rinse thoroughly with water for 15 minutes and seek medical advice if necessary. For research use only; not for human therapeutic use.
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| References | |
| Additional Infomation |
TNF-alpha-IN-6 is not an approved drug and is not in clinical development. It is a research-grade small molecule that serves as a valuable tool for investigating the biology of TNFalpha and its role in inflammatory diseases. Its unique allosteric mechanism of action, binding to TNFalpha with a KD of 6.8 nM, provides a distinct pharmacological profile compared to conventional TNFalpha inhibitors. It is used in pharmacological studies to explore new strategies for modulating TNFalpha signaling, which could potentially lead to the development of novel oral therapies for conditions such as rheumatoid arthritis, inflammatory bowel disease (IBD), and psoriasis. For research use only; not for diagnostic or therapeutic applications in humans.
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| Molecular Formula |
C26H25N9O2
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| Molecular Weight |
495.54
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| Exact Mass |
495.213
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| CAS # |
2699704-20-4
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| PubChem CID |
162641389
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| Appearance |
Light yellow to yellow solid powder
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| LogP |
1.3
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
9
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
37
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| Complexity |
942
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| Defined Atom Stereocenter Count |
1
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| SMILES |
C1(C#N)=NC=CC=C1CN1C2=NC(C3=CN=C(N4CCN5C(=O)NC[C@]5([H])C4)N=C3)=CC=C2C(=O)C1(C)C
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| InChi Key |
RUVSMFOFQJDXHE-GOSISDBHSA-N
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| InChi Code |
InChI=1S/C26H25N9O2/c1-26(2)22(36)19-5-6-20(32-23(19)35(26)14-16-4-3-7-28-21(16)10-27)17-11-29-24(30-12-17)33-8-9-34-18(15-33)13-31-25(34)37/h3-7,11-12,18H,8-9,13-15H2,1-2H3,(H,31,37)/t18-/m1/s1
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| Chemical Name |
3-[[6-[2-[(8aR)-3-oxo-1,2,5,6,8,8a-hexahydroimidazo[1,5-a]pyrazin-7-yl]pyrimidin-5-yl]-2,2-dimethyl-3-oxopyrrolo[2,3-b]pyridin-1-yl]methyl]pyridine-2-carbonitrile
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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: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light. |
| 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 (201.80 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.05 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.0180 mL | 10.0900 mL | 20.1800 mL | |
| 5 mM | 0.4036 mL | 2.0180 mL | 4.0360 mL | |
| 10 mM | 0.2018 mL | 1.0090 mL | 2.0180 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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