| Targets |
The parent compound, 2-Acetamidophenol (orthocetamol), has recently been identified as a regulator of ferroptosis, a form of regulated cell death driven by iron-dependent lipid peroxidation. It targets the glutathione (GSH) metabolic pathway and the cellular antioxidant system. Specifically, it enhances the activity of the key anti-ferroptotic enzyme, glutathione peroxidase 4 (GPX4). It acts by up-regulating the expression of genes involved in GSH synthesis, such as GCLC and GCLM, and iron transport genes (FPN1, FTH). The deuterated compound has the same target profile as the parent compound but is used primarily for analytical tracing.
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| ln Vitro |
Stable heavy isotopes of hydrogen, carbon, and other elements have been incorporated into drug molecules, largely as tracers for quantitation during the drug development process. Studies involving the human use of drugs labeled with deuterium suggest that these compounds may offer some advantages when compared with their nondeuterated counterparts. Deuteration has gained attention because of its potential to affect the pharmacokinetic and metabolic profiles of drugs. Deutetrabenazine is the first deuterated drug to receive Food and Drug Administration approval. This deuterated form of the drug tetrabenazine is indicated for the treatment of chorea associated with Huntington's disease as well as tardive dyskinesia. Ongoing clinical trials suggest that a number of other deuterated compounds are being evaluated for the treatment of human diseases and not merely as research tools.
In vitro, the parent compound 2-Acetamidophenol has demonstrated significant biological activity in models of ferroptosis. It lowers intracellular reactive oxygen species (ROS) and ferrous ion (Fe2+) accumulation, thereby preventing lipid peroxidation and cell death. It has also been shown to inhibit the phagocytosis of oxidized low-density lipoprotein (ox-LDL) by macrophages and the subsequent formation of foam cells, indicating anti-atherosclerotic activity. The deuterated version, 2-Acetamidophenol-d3, is inactive as a "drug" in bioassays but is chemically identical and used as an internal standard to precisely measure the parent compound in biological samples. |
| ln Vivo |
Deuterated compounds may, in some cases, offer advantages over nondeuterated forms, often through alterations in clearance. Deuteration may also redirect metabolic pathways in directions that reduce toxicities. The approval of additional deuterated compounds may soon follow. Clinicians will need to be familiar with the dosing, efficacy, potential side effects, and unique metabolic profiles of these new entities.
The in vivo activity of 2-Acetamidophenol itself has been explored in models of atherosclerosis and hyperlipidemia. It has been shown to lower total cholesterol (TC) and triglycerides (TG) in a zebrafish hyperlipidemia model, with IC50 values of 30 uM and 40 uM, respectively. However, the in vivo activity of the labeled version is not studied. Instead, the labeled compound is used as a tracer to understand the metabolism and pharmacokinetics of the parent compound. For its intended use as an analytical standard, it has no direct in vivo activity. |
| Enzyme Assay |
General in vitro anti-ferroptosis assay (cell-based): HT-1080 fibrosarcoma cells are seeded in 96-well plates and treated with a ferroptosis-inducing agent (e.g., RSL3, 0.1 uM). The cells are co-treated with 2-Acetamidophenol (0.1-100 uM) as the test compound, while the deuterated version is used as an internal standard for an associated LC-MS analysis. Cell viability is measured after 24 hours using the CellTiter-Glo® assay. A dose-dependent rescue of cell viability by the parent compound indicates inhibition of ferroptosis. Cytotoxicity can be assessed in parallel.
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| Cell Assay |
General in vitro lipid peroxidation assay (C11-BODIPY): Cells are treated with a ferroptosis inducer in the presence or absence of 2-Acetamidophenol. After 6-8 hours, cells are stained with the fluorescent lipid peroxidation sensor C11-BODIPY (1 uM). The fluorescence signal shifts from red to green when the dye is oxidized. The mean fluorescence intensity in the green channel is measured by flow cytometry or a fluorescence plate reader. The parent compound will prevent the shift to green, indicating reduced lipid peroxidation.
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| Animal Protocol |
General in vivo animal protocol for the parent compound: For the parent compound, a 14-day oral toxicity study in rats at 0, 100, 300, 1000 mg/kg/day is required. For the deuterated compound, it is not administered alone. It is used as an internal standard. For impurity qualification, it is not considered a genotoxic impurity. It is an isotopologue of a known molecule, and the safety of the parent compound would dictate its impurity limit. In a drug substance, it would be controlled at 0.15%.
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| ADME/Pharmacokinetics |
2-Acetamidophenol-d3 (MW 154.18) has the same ADME properties as the unlabeled parent compound. It has moderate lipophilicity (logP ~1.5). It is well-absorbed from the GI tract (oral bioavailability > 70%). It undergoes first-pass metabolism, primarily by glucuronidation and sulfation, and is rapidly cleared from the plasma (t½ ~2-3 h). The volume of distribution is moderate (~1-2 L/kg). It is not expected to cross the BBB. The deuterated version is an ideal isotopologue for tracing these pathways.
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| Toxicity/Toxicokinetics |
The parent compound, orthocetamol, is structurally similar to paracetamol but has not been as widely studied. It is generally considered to have a lower hepatotoxicity profile than paracetamol, but standard safety evaluations are not available. The deuterated compound is non-toxic at the minute concentrations used for bioanalysis. For its use as a reference standard, it is handled as a standard hazardous chemical. It is not classified as a genotoxic impurity.
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| References |
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| Additional Infomation |
Appearance: White solid. Molecular formula: C₈H₆D3NO2. Storage: -20degC. Solubility: Soluble in DMSO and ethanol. Other names: Orthocetamol-d3, N-(2-hydroxyphenyl)acetamide-d3. Safety: For research use only; avoid inhalation and skin contact.
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| Molecular Formula |
C8H6D3NO2
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|---|---|
| Molecular Weight |
154.18
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| CAS # |
122258-87-1
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| Related CAS # |
2-Acetamidophenol-d3
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| Appearance |
Powder
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| Synonyms |
Orthocetamol-d3
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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 (~648.59 mM; with sonication)
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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 | 6.4859 mL | 32.4296 mL | 64.8593 mL | |
| 5 mM | 1.2972 mL | 6.4859 mL | 12.9719 mL | |
| 10 mM | 0.6486 mL | 3.2430 mL | 6.4859 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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