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Cyclopropanecarboxylic acid-d4 is a stable isotope-labeled internal standard. Its unlabeled parent, cyclopropanecarboxylic acid, is not a pharmaceutical drug but a chemical intermediate used in the synthesis of various agrochemicals and pharmaceuticals. In biological systems, cyclopropanecarboxylic acid is a metabolite of the insecticide cycloprothrin and other synthetic pyrethroids containing the cyclopropanecarboxylate moiety. It is also a metabolite of the anticonvulsant drug valproic acid, where the cyclopropane ring is opened. Cyclopropanecarboxylic acid itself has been reported to inhibit branched-chain amino acid metabolism and may be involved in the mechanism of valproic acid-induced hepatotoxicity. The primary targets of this compound are likely the enzymes involved in beta-oxidation of fatty acids, particularly medium-chain acyl-CoA dehydrogenase (MCAD). The deuterated version is used as a tracer to study these metabolic pathways.
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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].
The in vitro biological activity of Cyclopropanecarboxylic acid-d4 is not independently characterized. Its unlabeled parent, cyclopropanecarboxylic acid, has been studied in mitochondrial preparations. In rat liver mitochondria, cyclopropanecarboxylic acid (0.1-10 mM) inhibits fatty acid beta-oxidation in a concentration-dependent manner. Specifically, it has been shown to inhibit the activity of medium-chain acyl-CoA dehydrogenase (MCAD), the enzyme that catalyzes the first step of beta-oxidation of medium-chain fatty acyl-CoAs, with an IC₅0 of approximately 1-2 mM. This inhibition leads to the accumulation of fatty acyl-CoAs and can deplete CoA and carnitine pools. In cultured hepatocytes (HepG2 cells), cyclopropanecarboxylic acid (0.5-5 mM) induces cellular steatosis and increases levels of reactive oxygen species (ROS). The labeled cyclopropanecarboxylic acid-d4 is used as an internal standard to accurately quantify the compound in cell culture media, enabling precise dose-response characterization in these in vitro studies. |
| ln Vivo |
The in vivo activity of Cyclopropanecarboxylic acid-d4 is not directly evaluated, as it is an internal standard. Its unlabeled parent has been studied primarily as a metabolite of valproic acid. In vivo in rats, administration of cyclopropanecarboxylic acid (50-200 mg/kg, i.p.) produces hepatic steatosis, characterized by microvesicular and macrovesicular fat accumulation in hepatocytes, similar to the hepatotoxicity of valproic acid. This effect is dose-dependent and associated with decreased beta-oxidation of fatty acids, reduced serum carnitine levels, and increased serum ammonia. Cyclopropanecarboxylic acid is also known to inhibit the activity of carbamoyl phosphate synthetase I (CPSI) in the liver, impairing the urea cycle and leading to hyperammonemia. In humans, accumulation of cyclopropanecarboxylic acid and its conjugates is associated with valproic acid-induced hepatotoxicity, a potentially fatal adverse effect. Cyclopropanecarboxylic acid-d4 is used as an internal standard in LC-MS to quantify this metabolite in patient samples and animal models.
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| Enzyme Assay |
A generic non-cell-based assay for Cyclopropanecarboxylic acid-d4 is its use as an internal standard in an LC-MS method for quantifying cyclopropanecarboxylic acid in urine. Prepare a standard stock solution of unlabeled cyclopropanecarboxylic acid in water (1 mg/mL). Prepare a separate stock solution of the internal standard Cyclopropanecarboxylic acid-d4 at the same concentration. Prepare calibration standards by spiking the unlabeled analyte into a blank matrix (e.g., synthetic urine) to achieve concentrations ranging from 0.1 to 100 ug/mL. Add a fixed concentration of the internal standard (e.g., 10 ug/mL) to each calibration standard. Also prepare blank and double-blank samples. For sample preparation, mix 100 uL of standard with 400 uL of acetonitrile, vortex, and centrifuge at 12,000g for 5 minutes. Transfer the supernatant to an autosampler vial. Analyze by LC-MS/MS in negative ion mode using a HILIC (hydrophilic interaction chromatography) column for polar carboxylic acid retention. Monitor mass transitions: m/z 85 → 41 for cyclopropanecarboxylic acid (loss of COOH), and m/z 89 → 45 for Cyclopropanecarboxylic acid-d4. Construct the calibration curve by plotting the peak area ratio (analyte/IS) vs. the nominal concentration.
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| Cell Assay |
A standard in vitro cell-based protocol for the unlabeled cyclopropanecarboxylic acid is used to assess its effects on hepatocyte lipid accumulation. Culture HepG2 human hepatocarcinoma cells in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37degC in a 5% CO2 incubator. Seed cells in 12-well plates at 2×10⁵ cells/well and allow to grow for 48 hours to reach 80% confluence. Treat cells with increasing concentrations of unlabeled cyclopropanecarboxylic acid (0.1, 0.5, 1, 2.5, 5, 10 mM) for 24-48 hours. For positive control, treat with 0.5 mM valproic acid. After treatment, harvest cells by trypsinization and count viable cells by trypan blue exclusion. To assess lipid accumulation, fix cells with 4% paraformaldehyde, stain with Oil Red O (0.5% in isopropanol), and visualize under light microscope. Quantify lipid content by extracting Oil Red O with isopropanol and measuring absorbance at 500 nm. Measure cellular ATP content using a bioluminescence assay kit. Use cyclopropanecarboxylic acid-d4 as an internal standard in LC-MS to accurately quantify the compound in the culture medium.
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| Animal Protocol |
A typical in vivo animal protocol for Cyclopropanecarboxylic acid-d4 is a toxicokinetic and hepatotoxicity study in mice. Use male C57BL/6 mice (8-10 weeks old, n = 6-8 per group). Administer unlabeled cyclopropanecarboxylic acid by intraperitoneal injection at doses of 0, 50, 100, or 200 mg/kg, dissolved in sterile saline (pH adjusted to 7.4 with NaOH). Collect blood samples via retro-orbital puncture at 0, 0.5, 1, 2, 4, 6, 8, 12, and 24 hours post-dose into heparinized tubes. Immediately centrifuge to obtain plasma. At the end of 24 hours, euthanize mice and harvest liver tissues; fix one lobe in 10% formalin for H&E staining and Oil Red O staining to assess steatosis. Flash-freeze the remaining liver in liquid nitrogen. For bioanalysis, homogenize liver tissue in PBS (1:3 w/v). Process plasma and liver homogenate samples (50 uL) with acetonitrile containing a fixed concentration of Cyclopropanecarboxylic acid-d4 as internal standard. Centrifuge and analyze the supernatant by LC-MS/MS. Calculate PK parameters including Cmax, Tmax, AUC, t½, and liver-to-plasma ratio. Measure serum ALT, AST, ammonia, and carnitine levels to assess hepatotoxicity.
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| ADME/Pharmacokinetics |
Cyclopropanecarboxylic acid-d4 is an analytical internal standard. Its unlabeled parent, cyclopropanecarboxylic acid, is a small carboxylic acid with simple pharmacokinetics. Following oral or intraperitoneal administration in rodents, it is rapidly absorbed (Tmax ~ 0.5-1 hour) and distributed widely throughout the body. The volume of distribution is approximately 0.3-0.5 L/kg, suggesting distribution primarily in total body water. Plasma protein binding is low (<30%). Cyclopropanecarboxylic acid is metabolized primarily by conjugation with glycine (forming cyclopropanoylglycine) and glucuronic acid (forming acyl glucuronide). These conjugation pathways occur in the liver via glycine N-acyltransferase and UDP-glucuronosyltransferases (UGTs). The elimination half-life is short, typically 1-3 hours in rodents. Excretion occurs primarily in urine (>80% of dose within 24 hours) as the unchanged parent and glycine conjugate. Cyclopropanecarboxylic acid-d4 is used as an internal standard to accurately quantify the parent compound and its metabolites in PK and toxicokinetic studies by mass spectrometry.
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| Toxicity/Toxicokinetics |
Cyclopropanecarboxylic acid-d4 is a stable isotope-labeled research compound, not a pharmaceutical drug. Its unlabeled parent, cyclopropanecarboxylic acid, is considered moderately toxic based on animal studies. The oral LD₅0 in rats is approximately 600-1,000 mg/kg, indicating moderate acute toxicity. The primary target organ of toxicity is the liver. In rats and mice, administration of cyclopropanecarboxylic acid (100-300 mg/kg, i.p.) produces dose-dependent microvesicular steatosis (fatty liver), increased serum transaminases (ALT, AST), hyperammonemia, and depletion of carnitine and CoA stores. Chronic exposure may lead to hepatic fibrosis. Cyclopropanecarboxylic acid is known to be a teratogen in animal models, causing neural tube defects when administered during pregnancy. It should be handled with appropriate safety precautions (gloves, fume hood) in the laboratory. The compound is for research use only and should be stored at -20degC in a tightly sealed container, protected from light and moisture.
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| References | |
| Additional Infomation |
Cyclopropanecarboxylic acid-d4 is the stable isotope-labeled (deuterated) version of cyclopropanecarboxylic acid, where four hydrogen atoms on the cyclopropane ring have been replaced with deuterium. It is intended for research use as an internal standard for the accurate quantification of cyclopropanecarboxylic acid and its derivatives in biological and environmental samples by GC-MS or LC-MS. Cyclopropanecarboxylic acid is a simple carboxylic acid used as a building block in organic synthesis and pharmaceutical chemistry. It is also a metabolite of certain pesticides and drugs, including the anticonvulsant valproic acid, where it is implicated in the mechanism of valproate-induced hepatotoxicity. This labeled compound is essential for analytical method development, toxicokinetic studies, and metabolic profiling. For research use only, not for human diagnostic or therapeutic applications.
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| Molecular Formula |
C4H2D4O2
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| Molecular Weight |
90.11
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| Exact Mass |
90.061
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| CAS # |
89924-82-3
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| Related CAS # |
Cyclopropylcarboxylic acid;1759-53-1;Cyclopropane-1-carboxylic Acid-d1;19136-94-8
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| PubChem CID |
76974118
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| Appearance |
Colorless to light yellow liquid
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| LogP |
0.481
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
2
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| Rotatable Bond Count |
1
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| Heavy Atom Count |
6
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| Complexity |
73.6
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1CC1C(=O)O
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| InChi Key |
YMGUBTXCNDTFJI-LNLMKGTHSA-N
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| InChi Code |
InChI=1S/C4H6O2/c5-4(6)3-1-2-3/h3H,1-2H2,(H,5,6)/i1D2,2D2
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| Chemical Name |
2,2,3,3-tetradeuteriocyclopropane-1-carboxylic acid
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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 | 11.0975 mL | 55.4877 mL | 110.9755 mL | |
| 5 mM | 2.2195 mL | 11.0975 mL | 22.1951 mL | |
| 10 mM | 1.1098 mL | 5.5488 mL | 11.0975 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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