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| Targets |
Unlike its parent drug 5-FU, 5,6-Dihydro-5-fluorouracil itself does not directly inhibit thymidylate synthase (TS) or incorporate into nucleic acids to exert antitumor activity. Instead, it functions as the metabolic product of 5-FU catabolism via the enzyme dihydropyrimidine dehydrogenase (DPYD, EC 1.3.1.2), which catalyzes the reduction of 5-FU to 5-FUH2 using NADPH as a cofactor . The compound is further hydrolyzed by dihydropyrimidinase (DPYS) to α-fluoro-β-ureidopropionic acid, representing an inactivation step that limits the availability and cytotoxic effects of 5-FU .
5,6-Dihydro-5-Fluorouracil targets the same pathways as its parent compound 5-fluorouracil. As a metabolite formed by DPD, it is part of the 5-FU metabolic pathway. The compound is cytotoxic to cells, with demonstrated activity against HaCaT keratinocytes. Its formation is a key step in 5-FU metabolism and clearance. |
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| ln Vitro |
In vitro studies have demonstrated that 5,6-Dihydro-5-fluorouracil itself exhibits minimal direct cytotoxic activity against cancer cell lines. However, research on structurally related 5-fluoro-5,6-dihydro-6-alkoxy-uracil derivatives has shown that certain analogues with a cis-configuration, particularly cis-5-F-5,6-dihydro-6-methoxy-uracil, exert more potent antiproliferative effects than 5-FU itself in some solid tumor cell lines, with marked inhibition of thymidylate synthase as measured by tritiated deoxyuridine incorporation assays . The catalytic efficiency of dihydropyrimidine dehydrogenase for oxidizing R-FUH2 is only 1/14th of that for the natural substrate 5,6-dihydrouracil .
In vitro, 5,6-Dihydro-5-Fluorouracil is cytotoxic to HaCaT keratinocytes with an IC50 of 13.5 μM. It is the active metabolite formed from 5-fluorouracil by dihydropyrimidine dehydrogenase (DPD). Its cytotoxicity contributes to the overall pharmacological effects of 5-fluorouracil therapy. |
| ln Vivo |
In vivo studies have shown that 5,6-Dihydro-5-fluorouracil attenuates the antitumor activity of 5-FU. In a rat model bearing large subcutaneous tumors, combining 5-FU with 90 mg/kg 5-FUH2 (to approximate the catabolite exposure of 5-FU alone) reduced the complete tumor regression rate from 94% (with 5-EU pretreatment preventing catabolite formation) to 38%, comparable to 5-FU treatment without catabolite prevention. Additionally, 5-FUH2 contributed to 5-FU-associated toxicities including transient diarrhea and stomatitis in 13% of animals . In clinical studies, plasma concentrations of 5-FUH2 ranged from 131.98 to 987.93 ng/mL in colorectal cancer patients receiving 5-FU-based chemotherapy, with significant correlation between 5-FUH2 levels and adverse reactions .
In vivo, intravenous administration of 5,6-Dihydro-5-Fluorouracil (90 mg/kg/week) in combination with 5-fluorouracil and the DPD inhibitor eniluracil slows tumor growth in a rat colon cancer model. This demonstrates that the metabolite contributes to the antitumor activity of 5-FU, and that DPD inhibition can modulate its formation and effects. |
| Enzyme Assay |
For assessing dihydropyrimidine dehydrogenase (DPD) activity and 5-FU catabolism, a validated HPLC method with diode array detection has been developed. The protocol involves extracting 5-FU and 5-FUH2 from biological matrices (plasma or cellular lysates) using sodium acetate, sodium sulfate, and diethyl ether/propanol extraction. Dried samples are reconstituted in a mobile phase (35 mmol/L KH₂PO₄, pH 4.0) and isocratically eluted using a Hypersil C18 stationary phase (25 cm × 4.6 mm, 10 μm) with detection at 215 nm (reference 360 nm). 5-Fluorocytosine serves as the internal standard. Recovery rates range from 81-85%, with limits of detection and quantification of 3.2 and 16 ng/mL respectively, and both within-day and between-day CV <10% .
The non-cellular assay for 5,6-Dihydro-5-Fluorouracil involves characterizing its chemical properties. Key analytical techniques include HPLC, NMR, and mass spectrometry to confirm structure and purity. DPD enzyme activity can be assessed by measuring the conversion of 5-fluorouracil to 5,6-dihydro-5-fluorouracil in cell-free systems. |
| Cell Assay |
For cellular assays evaluating the effects of 5,6-Dihydro-5-fluorouracil, cell lines (e.g., various solid tumor cell lines) are cultured in appropriate medium. The test compound is added at varying concentrations, and antiproliferative activity is assessed using cell counting or colorimetric assays such as MTT. For thymidylate synthase inhibition studies in intact cells, incorporation of tritiated deoxyuridine ([³H]-UdR) into DNA is measured after exposure to the compound, and thymidine rescue experiments are performed to confirm TS as the mechanism of action. The cis-configured analogues have demonstrated higher activity than trans-configured derivatives in these cellular models .
In vitro cell-based assays for 5,6-Dihydro-5-Fluorouracil involve culturing cells such as HaCaT keratinocytes. Cells are treated with varying concentrations of the compound, and cytotoxicity is assessed using standard assays such as MTT or CellTiter-Glo to determine IC50 values. |
| Animal Protocol |
A representative in vivo protocol from a rat tumor model: Rats bearing large subcutaneous tumors are dosed once weekly for 3 weeks. The treatment regimen includes 5-EU (1 mg/kg) administered 1 hour prior to 5-FU (10 mg/kg) with or without 5-FUH2 (90 mg/kg). Tumor volumes are measured regularly, and body weight is monitored as an indicator of toxicity. Antitumor activity is assessed by the frequency of complete and sustained tumor regressions. Diarrhea and stomatitis are recorded as toxicity endpoints. In this model, 5-FU alone (100 mg/kg, maximum tolerated dose) and 5-FU/5-EU combinations are compared to evaluate the impact of 5-FUH2 on therapeutic outcomes .
In vivo animal study protocols for 5,6-Dihydro-5-Fluorouracil involve rat colon cancer models. The compound is administered intravenously at 90 mg/kg/week in combination with 5-fluorouracil and the DPD inhibitor eniluracil. Endpoints include tumor growth inhibition and assessment of drug metabolism. |
| ADME/Pharmacokinetics |
In cancer patients receiving 5-FU-based chemotherapy, plasma concentrations of 5,6-Dihydro-5-fluorouracil ranged from 131.98 to 987.93 ng/mL (mean 550.58 ± 260.60 ng/mL) . The compound is formed rapidly from 5-FU via dihydropyrimidine dehydrogenase, which catalyzes the reduction of 5-FU to 5-FUH2 with a Km of 0.70 μM and a kcat of 3 sec⁻¹ for the reductive direction. The subsequent hydrolysis of R-FUH2 by dihydropyrimidinase is highly efficient, with a Km of 130 μM and a kcat of 126 sec⁻¹. Because DPHase activity is 250- to 500-fold greater than DPDase activity in rat and bovine liver, the hydrolytic pathway (conversion to α-fluoro-β-ureidopropionic acid) predominates in vivo . The elimination half-life of 5-FUH2 is longer than that of 5-FU, contributing to its accumulation and clinical impact on both efficacy and toxicity .
Pharmacokinetic properties of 5,6-Dihydro-5-Fluorouracil are related to its formation as a metabolite of 5-fluorouracil. It is formed by DPD and its levels reflect DPD activity. The compound has a molecular weight of 132.09. Its formation and clearance are important for understanding 5-FU pharmacology and toxicity. |
| Toxicity/Toxicokinetics |
5,6-Dihydro-5-fluorouracil is a major catabolite of 5-FU and contributes to both the attenuation of antitumor efficacy and the development of adverse reactions. In clinical studies, a significant correlation exists between 5-FUH2 plasma concentrations and adverse reactions in colorectal cancer patients receiving 5-FU-based chemotherapy . In animal studies, addition of 5-FUH2 to 5-FU regimens caused transient diarrhea and stomatitis in 13% of treated animals, toxicities similar to those produced by 5-FU alone in the absence of catabolite prevention . As a research compound, 5-FUH2 is strictly intended for laboratory use only. General safety precautions include standard laboratory handling procedures; no specific acute toxicity (LD50) data is available in the provided references.
Toxicological data for 5,6-Dihydro-5-Fluorouracil are related to its role as a 5-FU metabolite. It is cytotoxic to cells and contributes to the toxicity profile of 5-fluorouracil. DPD deficiency can lead to accumulation of 5-FU and its metabolites, resulting in severe toxicity. Standard safety precautions should be observed. |
| References | |
| Additional Infomation |
5,6-Dihydro-5-fluorouracil is a pyrimidinone. It is a metabolite of 5-fluorouracil; the reference number given refers to the parent compound.
5,6-Dihydro-5-Fluorouracil (5-DHFU) is the active metabolite of 5-fluorouracil formed by dihydropyrimidine dehydrogenase (DPD). It is cytotoxic and contributes to the antitumor activity of 5-FU. It is a research compound used to study 5-FU metabolism and pharmacology and is not a therapeutic drug itself. |
| Molecular Formula |
C4H5FN2O2
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|---|---|
| Molecular Weight |
132.09
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| Exact Mass |
132.034
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| CAS # |
696-06-0
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| PubChem CID |
121997
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| Appearance |
White to off-white solids at room temperature
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| Density |
1.43g/cm3
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| Melting Point |
228-230ºC
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| Index of Refraction |
1.48
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| LogP |
0
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
0
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| Heavy Atom Count |
9
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| Complexity |
159
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| Defined Atom Stereocenter Count |
0
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| SMILES |
FC1=CNC(=O)NC1=O
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| InChi Key |
RAIRJKWTBBDDAR-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C4H5FN2O2/c5-2-1-6-4(9)7-3(2)8/h2H,1H2,(H2,6,7,8,9)
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| Chemical Name |
5-fluoro-1,3-diazinane-2,4-dione
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| Synonyms |
5-DHFU; 5-Fluorodihydropyrimidine-2,4-dione; 5-fluorodihydrouracil; 5,6-Dihydro-5-fluorouracil; DHFU; 5,6-Dihydrofluorouracil; 5-Fluorodihydrouracil
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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 | 7.5706 mL | 37.8530 mL | 75.7060 mL | |
| 5 mM | 1.5141 mL | 7.5706 mL | 15.1412 mL | |
| 10 mM | 0.7571 mL | 3.7853 mL | 7.5706 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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