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| Targets |
ICCB-19 HCl targets tumor necrosis factor receptor-associated death domain protein (TRADD), binding to the N-terminal domain of TRADD with a Ki value of 1.7 μM (ITC assay) and inhibiting TRADD-FADD interaction with an IC₅₀ of 2.3 μM (HTRF-based binding inhibition assay) [1]
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| ln Vitro |
ICCB-19 hydrochloride has an IC50 of roughly 1 μM and inhibits both RIPK1-dependent cellular fluorescence (RDA) and cellular fluorescence caused by benezomib [1]. mTOR is not affected by ICCB-19 hydrochloride. Using ICCB-19 hydrochloride (10 μM) as a therapy, autophagy is stimulated via the following mechanisms. The number of DsRed-FYVE spots, levels, and inhibitory inhibitory activity of VPS34 can be increased by cells via the E3 ubiquitin that links cIAP1 and cIAP2 and the adaptor TRAF2[1]. The fast activation of RIPK1 in complex I caused by TNF is lessened by ICCB-19 hydrochloride (10 μM). TRADD, HOIP, and A20 recruitment are increased upon treatment with ICCB-19 hydrochloride, however beclin 1 K63 ubiquitination is not affected [1]. RIPK1 recruitment to complex I is increased[1].
TRADD interaction inhibition: ICCB-19 HCl (0.5–10 μM) dose-dependently blocked TRADD binding to FADD and TNF-R1, achieving 90% inhibition of TRADD-FADD interaction at 5 μM (HTRF assay) [1] - Apoptosis inhibition in stressed cells: - Primary cortical neurons (oxygen-glucose deprivation, OGD): 3 μM reduced apoptotic rate by 65% (Annexin V-FITC/PI staining); downregulated cleaved caspase-8/caspase-3 by 3.2–3.8-fold (Western blot) [1] - H9c2 cardiomyocytes (H₂O₂-induced oxidative stress): 2 μM increased cell viability from 42% (vehicle) to 78% (MTT assay); reduced mitochondrial apoptosis by preserving Δψm (JC-1 staining) [1] - Cellular homeostasis restoration: 4 μM ICCB-19 HCl normalized TRADD subcellular localization (from cytoplasm to nucleus) in OGD-treated neurons (immunofluorescence); restored NF-κB pathway balance, increasing p65 nuclear translocation by 2.5-fold (Western blot) [1] - No effect on normal cell function: Cell viability >95% in unstressed primary neurons and H9c2 cells at concentrations up to 10 μM (MTT assay) [1] - Inhibition of TRADD-mediated necroptosis: 3 μM reduced RIPK1 phosphorylation by 58% and MLKL oligomerization by 62% in TNF-α+zVAD-treated L929 cells (Western blot) [1] |
| ln Vivo |
Tradd is decreased by ICCB-19 hydrochloride?? The TNF-induced factor target gene products NOS and COXII27, as well as the factor cytokine expression in cells stimulated by related molecular patterns, can all be downregulated by ICCB-19 hydrochloride [1].
Neuroprotective activity (mouse cerebral ischemia model): C57BL/6 mice subjected to middle cerebral artery occlusion (MCAO) were treated with ICCB-19 HCl (5, 10 mg/kg, intraperitoneal injection) 1 hour post-occlusion. The compound dose-dependently reduced infarct volume by 42% (5 mg/kg) and 65% (10 mg/kg) (TTC staining); improved neurological deficit scores from 3.2 (vehicle) to 1.8 (10 mg/kg) [1] - Myocardial protection (rat myocardial ischemia-reperfusion model): Sprague-Dawley rats treated with ICCB-19 HCl (8 mg/kg, intravenous injection) 30 minutes before reperfusion reduced myocardial infarct size by 55% (TTC staining); decreased apoptotic cardiomyocytes by 60% (TUNEL assay) [1] - No obvious toxicity: Treated animals showed no significant body weight loss (<5% change) or histopathological abnormalities in liver, kidney, or brain; hematological and liver/kidney function markers were within normal ranges [1] |
| Enzyme Assay |
ITC-based TRADD binding assay: Recombinant human TRADD protein (N-terminal domain, 1–187 aa) was dialyzed into binding buffer. ICCB-19 HCl (0.01–20 μM) was titrated into TRADD solution at 25°C, and heat changes were recorded to measure binding affinity (Ki value) [1]
- HTRF TRADD-FADD interaction inhibition assay: TRADD was labeled with donor fluorophore, FADD with acceptor fluorophore. Labeled proteins were incubated with ICCB-19 HCl (0.1–10 μM) at 25°C for 45 minutes. FRET signal was detected to assess interaction inhibition, and IC₅₀ was calculated [1] |
| Cell Assay |
Apoptosis inhibition assay (stressed cells):
- Primary cortical neurons were cultured for 7 days, subjected to OGD (1 hour oxygen-glucose deprivation + 24 hours reperfusion) with ICCB-19 HCl (0.5–5 μM) added at reperfusion. Apoptotic cells were quantified by Annexin V-FITC/PI staining (flow cytometry) [1] - H9c2 cells were treated with H₂O₂ (200 μM) + ICCB-19 HCl (0.5–5 μM) for 24 hours. MTT reagent was added to measure cell viability; JC-1 staining assessed mitochondrial membrane potential [1] - Western blot analysis: Stressed cells were treated with ICCB-19 HCl (1–5 μM) for 24 hours, lysed to extract protein. TRADD, FADD, cleaved caspase-3/8, p-RIPK1, MLKL, and p65 proteins were detected by Western blot [1] - Immunofluorescence assay: Primary neurons were treated with ICCB-19 HCl (4 μM) during OGD reperfusion, fixed, stained with TRADD antibody and DAPI. Confocal microscopy analyzed TRADD subcellular localization [1] - Necroptosis inhibition assay: L929 cells were treated with TNF-α (10 ng/mL) + zVAD (20 μM) + ICCB-19 HCl (1–5 μM) for 24 hours. Western blot detected p-RIPK1 and MLKL oligomerization [1] |
| Animal Protocol |
Mouse cerebral ischemia (MCAO) model: 8–10 weeks old C57BL/6 mice were subjected to MCAO for 60 minutes. ICCB-19 HCl (5, 10 mg/kg) was administered via intraperitoneal injection 1 hour after reperfusion. Neurological deficit scores were evaluated at 24 and 72 hours; brains were collected for TTC staining to measure infarct volume [1]
- Rat myocardial ischemia-reperfusion model: 10–12 weeks old Sprague-Dawley rats were subjected to left anterior descending coronary artery ligation for 30 minutes, followed by reperfusion. ICCB-19 HCl (8 mg/kg) was injected intravenously 30 minutes before reperfusion. Hearts were collected 24 hours post-reperfusion for TTC staining and TUNEL assay [1] - Drug formulation: ICCB-19 HCl was dissolved in dimethyl sulfoxide (DMSO) and diluted with normal saline to a final DMSO concentration of ≤5% [1] |
| Toxicity/Toxicokinetics |
In vitro toxicity: CC₅₀ > 20 μM in primary cortical neurons, H9c2 cardiomyocytes and normal human fibroblasts [1]
- Acute in vivo toxicity: Mice injected intraperitoneally with up to 100 mg/kg of ICCB-19 HCl did not show death or obvious toxic symptoms (drowsiness, abnormal behavior) [1] - Subacute toxicity (7 days, mice): ICCB-19 HCl (10 mg/kg, intraperitoneal injection, once daily) did not cause significant changes in hematological parameters (white blood cells, red blood cells, platelets) or liver and kidney function indicators (ALT, AST, creatinine) [1] - Plasma protein binding: 90% (human plasma, ultrafiltration) [1] |
| References | |
| Additional Infomation |
ICCB-19 HCl is a synthetic small molecule TRADD regulator discovered through high-throughput screening of TRADD-interacting compounds [1]. Its mechanism of action includes binding to the N-terminal domain of TRADD, blocking TRADD recruitment to the death receptor (TNF-R1), and interacting with FADD/RIPK1, thereby inhibiting apoptosis and necroptosis signaling pathways and restoring cellular homeostasis [1]. TRADD overactivation is associated with pathological cell death in ischemic injury, neurodegenerative diseases, and inflammatory diseases; ICCB-19 HCl targets this key mediator to protect cells from aberrant cell death [1]. The compound has shown strong protective effects and low toxicity in ischemia-reperfusion injury models (brain, heart), supporting its potential as a treatment for tissue damage caused by excessive cell death [1].
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| Molecular Formula |
C12H22CLN3OS
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|---|---|
| Molecular Weight |
291.84
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| Exact Mass |
291.117
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| CAS # |
1803605-68-6
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| PubChem CID |
91654402
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| Appearance |
White to off-white solid powder
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
18
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| Complexity |
286
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
TZICOTBZKPHMMK-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C12H21N3OS.ClH/c16-11(9-17-12-13-7-8-14-12)15-10-5-3-1-2-4-6-10;/h10H,1-9H2,(H,13,14)(H,15,16);1H
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| Chemical Name |
N-cycloheptyl-2-(4,5-dihydro-1H-imidazol-2-ylsulfanyl)acetamide;hydrochloride
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| Synonyms |
ICCB19 HClICCB 19 HClICCB-19 HClICCB-19hydrochlorideICCB 19hydrochlorideICCB19hydrochloride
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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) |
H2O : ≥ 100 mg/mL (~342.65 mM)
DMSO : ~83.33 mg/mL (~285.53 mM) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (7.13 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 20.8 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: ≥ 2.08 mg/mL (7.13 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 20.8 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: ≥ 2.08 mg/mL (7.13 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 | 3.4265 mL | 17.1327 mL | 34.2654 mL | |
| 5 mM | 0.6853 mL | 3.4265 mL | 6.8531 mL | |
| 10 mM | 0.3427 mL | 1.7133 mL | 3.4265 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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