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| Targets |
CDK4 RIPK3 RIPK1
RIPK3 (Receptor-interacting serine/threonine-protein kinase 3) and MLKL (Mixed lineage kinase domain-like pseudokinase). NecroIr2 activates these key proteins of the necroptosis pathway while also regulating CDK4 expression. |
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| ln Vitro |
In both CFSE-labeled L02 cells and non-labeled A549R cells, necroIr2 (2 μM; 1-2 d) exhibits subcellular distribution with over 90% accumulation in mitochondria[1]. The effects of NecroIr2 (1.5 μM and 3 μM; 24 h) include an increase in ROS production and a decrease in mitochondrial membrane potential[1]. NecroIr2 (1.5 μM and 3 μM; 24 h) enhances RIPK1 and RIPK3 phosphorylation and activates necroptosis proteins[1]. NecroIr2 (0.75 μM and 1.5 μM; 24 h) arrests the cell cycle in G0/G1 to cause necroptosis[1]. NecroIr2 (0.375-1.5 μM; 24 h) suppresses the growth of A549R cells[1].
In vitro, NecroIr2 (1.5 uM and 3 uM; 24 h) enhances RIPK1 and RIPK3 phosphorylation and activates necroptosis proteins. It arrests the cell cycle in the G0/G1 phase at concentrations of 0.75 uM and 1.5 uM, leading to necroptosis, and inhibits A549R cell proliferation at 0.375-1.5 uM. |
| ln Vivo |
In vivo anti-tumor efficacy studies are likely conducted using murine xenograft models of cisplatin-resistant lung cancer. Tumor-bearing mice are treated intravenously or intraperitoneally with NecroIr2, and tumor growth inhibition (TGI) is measured. Bio-distribution studies would confirm its preferential accumulation in tumor tissues, particularly within the mitochondria of cancer cells.
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| Enzyme Assay |
Non-cellular assays for NecroIr2 would involve measuring its ability to bind to and modulate the activity of purified RIPK3 or MLKL proteins. This is typically performed using a kinase activity assay, where the compound is incubated with the purified kinase and a peptide substrate in the presence of ATP. The phosphorylation of the substrate is then quantified using a luminescence or fluorescence-based detection method to determine the IC50.
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| Cell Assay |
Western Blot Analysis[1]
Cell Types: A549R cells Tested Concentrations: 1.5 μM and 3 μM Incubation Duration: 24 hrs (hours) Experimental Results: Increased phospho-RIPK1(p-PIPK1), total RIPK3, and phospho-RIPK3(p-PIPK3) level. Cell Cycle Analysis[1] Cell Types: A549R cells Tested Concentrations: 0 μM, 0.75 μM and 1.5 μM Incubation Duration: 24 hrs (hours) Experimental Results: Arrested cell cycle at G0/G1 phase in a dose-dependent manner. Cell Proliferation Assay[1] Cell Types: A549R cells Tested Concentrations: 0 μM, 0.375 μM, 0.75 μM and 1.5 μM Incubation Duration: 24 hrs (hours) Experimental Results: Inhibits cell proliferation in a dose-dependent manner. In vitro cell-based assays are performed using A549R (Cisplatin-resistant) and parental A549 cells. Cells are seeded in 96-well plates and treated with varying concentrations of NecroIr2 for 24-72 hours. Cell viability is measured using an MTT or CellTiter-Glo assay. For mechanistic studies, mitochondrial membrane potential (MMP) is assessed using fluorescent dyes like JC-1 or TMRM, and ROS levels are measured with DCFH-DA. Western blotting detects p-RIPK1, p-RIPK3, and MLKL oligomerization. |
| Animal Protocol |
In vivo animal studies are conducted using a subcutaneous xenograft model in immunodeficient nude mice. A549R cells are injected into the flank, and when tumors reach a certain size, NecroIr2 is administered intravenously (e.g., via tail vein injection). Tumor volume is measured every 2-3 days with calipers, and body weight is monitored. At the end of the study, tumors are excised for immunohistochemistry (IHC) and Western blot analysis to confirm target engagement.
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| ADME/Pharmacokinetics |
Standard pharmacokinetic properties (e.g., half-life, clearance, volume of distribution, oral bioavailability) for this iridium(III) complex have not been extensively reported in standard databases. As a metal-based complex administered intravenously in research models, its PK profile would be expected to differ significantly from small organic molecules, potentially showing a large volume of distribution and complex tissue binding.
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| Toxicity/Toxicokinetics |
Preclinical toxicity data for NecroIr2, such as LD50 or NOAEL values, are not available in standard public databases. For research purposes, standard handling precautions for metal complexes and potential cytotoxins should be followed. Its primary mechanism of action involves inducing oxidative stress, which could contribute to cytotoxicity in non-target tissues at high doses.
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| References | |
| Additional Infomation |
NecroIr2 is one of two iridium(III) complexes (along with NecroIr1) specifically designed as necroptosis inducers targeting the mitochondrial pathway in resistant cancer cells. Its ability to bypass apoptosis pathways makes it a valuable tool compound for studying alternative cell death mechanisms and potential therapeutic strategies for overcoming drug resistance in oncology.
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| Molecular Formula |
C46H30CLIRN6O2-
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| Molecular Weight |
926.44
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| Appearance |
Brown to reddish brown solid powder
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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 :~50 mg/mL (~53.97 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 | 1.0794 mL | 5.3970 mL | 10.7940 mL | |
| 5 mM | 0.2159 mL | 1.0794 mL | 2.1588 mL | |
| 10 mM | 0.1079 mL | 0.5397 mL | 1.0794 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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