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| Targets |
NNK (non-deuterated) is a procarcinogen that targets various tissues, particularly the lungs, liver, nasal mucosa, and pancreas. It is metabolically activated by cytochrome P450 enzymes (primarily CYP2A13 in the respiratory tract and CYP2A6 in the liver) via alpha-hydroxylation to form reactive intermediates that alkylate DNA, producing DNA adducts such as O6-methylguanine, which lead to activating mutations in oncogenes (e.g., K-ras) and inactivating mutations in tumor suppressor genes (e.g., p53). NNK also induces oxidative stress and inflammation, contributing to its carcinogenic effects. The deuterated version (NNK-d4) is not used for activity studies but serves as an internal standard.
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| ln Vitro |
Drug compounds have included stable heavy isotopes of carbon, hydrogen, and other elements, mostly as quantitative tracers while the drugs were being developed. Because deuteration may have an effect on a drug's pharmacokinetics and metabolic properties, it is a cause for concern [1].
In vitro, NNK (non-deuterated) is used in cell culture models to study tobacco carcinogen-induced DNA damage, mutagenesis, and malignant transformation. Treatment of human bronchial epithelial cells or lung cancer cell lines with NNK (1-100 uM) for 24-72 hours induces DNA adduct formation (measured by 32P-postlabeling or LC-MS), DNA strand breaks (comet assay), and mutations in the K-ras gene. NNK also activates various signaling pathways including MAPK/ERK, PI3K/Akt, and NF-kappaB, and induces oxidative stress (ROS production). NNK-d4 is not used in these activity studies; it is used as an internal standard for quantifying NNK and its metabolites in cell lysates and culture media. |
| ln Vivo |
In vivo, NNK (non-deuterated) is a potent pulmonary carcinogen in animal models (mice, rats, hamsters). In A/J mice, a single intraperitoneal injection of NNK (1-10 mg/kg) induces lung adenomas and adenocarcinomas within 16-20 weeks. In rats, NNK induces tumors of the lung, nasal cavity, liver, and pancreas. NNK also induces DNA adduct formation in target tissues, which can be measured by LC-MS/MS. NNK-d4 is not used for in vivo efficacy studies; it is used as an internal standard for the quantification of NNK and its metabolites in animal tissues and fluids for toxicokinetic studies and biomonitoring of tobacco exposure.
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| Enzyme Assay |
For non-cellular assays (analytical quantification), NNK-d4 is prepared as a stock solution in methanol or acetonitrile (1 mg/mL). For LC-MS/MS analysis, a calibration curve for NNK is prepared in human plasma, urine, or tobacco product extracts (0.1-1000 ng/mL) with a fixed concentration of NNK-d4 (e.g., 10-50 ng/mL). Sample preparation: 500 uL plasma or urine + 50 uL internal standard + 2 mL acetonitrile for protein precipitation. After centrifugation, the supernatant is evaporated to dryness, reconstituted in 200 uL mobile phase (0.1% formic acid in water and acetonitrile, 80:20, v/v), and injected onto a C18 column (2.1×50 mm, 1.8 um). MRM transitions: NNK 208→122 (and 208→79), NNK-d4 212→126 (and 212→79). For GC-MS analysis, NNK and NNK-d4 are derivatized with pentafluoropropionic anhydride (PFPA) before injection.
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| Cell Assay |
For cell-based assays, human lung epithelial cells (e.g., BEAS-2B normal bronchial epithelial cells, A549 lung adenocarcinoma cells) are seeded in 6-well plates (1×10⁶ cells/well) in DMEM with 10% FBS. Cells are treated with NNK (0.1-100 uM) or NNK-d4 (as a tracer) for 24-72 hours. DNA adducts (O6-methylguanine, 7-methylguanine) are measured by LC-MS/MS after DNA extraction and enzymatic hydrolysis. For mutation analysis, cells are cultured for 7-14 days after NNK treatment, and DNA is extracted for sequencing of K-ras and p53 genes. For genotoxicity studies, the comet assay is performed. For metabolic studies, NNK metabolism is assessed by measuring NNK and its metabolites (e.g., NNAL, NNAL-glucuronide, keto acid) in culture medium by LC-MS/MS using NNK-d4 as internal standard. Cell viability is assessed by MTT assay.
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| Animal Protocol |
For in vivo animal experiments, A/J mice (6-8 weeks old) are used for lung carcinogenicity studies. Mice are administered NNK intraperitoneally (2 mg/kg) once per week for 8 weeks or as a single dose (10 mg/kg). For biomonitoring studies, rodents are exposed to tobacco smoke or administered NNK (0.1-10 mg/kg), and urine and blood are collected at multiple time points. NNK-d4 is added as an internal standard to urine, plasma, or tissue homogenates before LC-MS/MS analysis to quantify NNK and its metabolites (e.g., NNAL, NNAL-glucuronide). For DNA adduct studies, lung and other target tissues are harvested at various time points (1 hour to 28 days post-dosing), DNA is extracted, and adducts are measured by LC-MS/MS or 32P-postlabeling. For toxicokinetic studies, NNK-d4 can be administered as a tracer.
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| ADME/Pharmacokinetics |
NNK-d4 has a molecular weight of 211.25, with deuterium atoms located at positions 3,4,5, and 6 of the pyridine ring. The compound is a light yellow to colorless oil or solid (depending on purity and temperature). It is soluble in organic solvents such as methanol, acetonitrile, DMSO, and chloroform. The compound is a nitrosamine, which is a class of chemicals that are potent carcinogens. It should be stored as a powder or solution at -20degC, protected from light. NNK is stable at low temperatures but may degrade upon prolonged exposure to light or heat. The deuterated version is chemically stable and non-radioactive. Due to the carcinogenic nature of NNK, extreme care must be taken when handling the non-deuterated compound, but NNK-d4 at analytical concentrations (ng-ug) poses minimal risk.
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| Toxicity/Toxicokinetics |
NNK-d4 is a stable isotope-labeled compound used as an internal standard for analytical quantification. The non-deuterated parent compound, NNK, is a potent tobacco-specific nitrosamine and a Group 1 human carcinogen (classified by IARC). It is highly toxic and carcinogenic; direct handling of non-deuterated NNK requires special precautions including use of a fume hood, double gloves, and containment. NNK-d4 itself at the low concentrations used as an internal standard (ng-ug per sample) poses minimal toxicity risk, but standard safety precautions for handling nitrosamines should be followed (use of PPE, work in fume hood, avoid inhalation, ingestion, and skin contact). The compound is non-radioactive.
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| References | |
| Additional Infomation |
See other relationships...
NNK-d4 is an analytical standard and research tool, not an approved drug. No clinical trials have been conducted with the deuterated version for therapeutic purposes. The non-deuterated parent compound, NNK, is a potent tobacco-specific nitrosamine carcinogen and is not used therapeutically. NNK-d4 is used exclusively as an internal standard for quantitative LC-MS/MS analysis of NNK and its metabolites in research and analytical settings, including biomonitoring of tobacco exposure, toxicokinetic studies, and environmental monitoring of tobacco products. It is also used in studies of nitrosamine metabolism, DNA adduct formation, and mechanisms of tobacco-related carcinogenesis. The compound is a valuable tool for assessing the carcinogenic potential of tobacco products and for evaluating interventions to reduce nitrosamine exposure. Available for research use only. Not intended for diagnostic or therapeutic use. |
| Molecular Formula |
C10H9D4N3O2
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|---|---|
| Molecular Weight |
211.25
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| Exact Mass |
211.126
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| CAS # |
764661-24-7
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| Related CAS # |
NNK;64091-91-4;NNK-d3;86270-92-0
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| PubChem CID |
12147167
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| Appearance |
White to off-white solid powder
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| Density |
1.18g/cm3
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| Boiling Point |
423.908ºC at 760 mmHg
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| Melting Point |
60-62ºC
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| Flash Point |
210.172ºC
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| Index of Refraction |
1.557
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| LogP |
1.657
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
15
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| Complexity |
221
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| Defined Atom Stereocenter Count |
0
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| SMILES |
[2H]C1=C(C(=C(N=C1[2H])[2H])C(=O)CCCN(C)N=O)[2H]
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| InChi Key |
FLAQQSHRLBFIEZ-MNYIHESISA-N
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| InChi Code |
InChI=1S/C10H13N3O2/c1-13(12-15)7-3-5-10(14)9-4-2-6-11-8-9/h2,4,6,8H,3,5,7H2,1H3/i2D,4D,6D,8D
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| Chemical Name |
N-methyl-N-[4-oxo-4-(2,4,5,6-tetradeuteriopyridin-3-yl)butyl]nitrous amide
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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) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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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 | 4.7337 mL | 23.6686 mL | 47.3373 mL | |
| 5 mM | 0.9467 mL | 4.7337 mL | 9.4675 mL | |
| 10 mM | 0.4734 mL | 2.3669 mL | 4.7337 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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