Endogenous Metabolite; Dihydrouracil is a substrate/product of the enzyme dihydropyrimidine dehydrogenase (DPD). The provided documents do not include IC50, Ki, or EC50 values for dihydrouracil as it is an endogenous metabolite, not a drug candidate targeting a specific protein. [1][2]
As an endogenous metabolite, 5,6-dihydrouracil itself does not have a specific pharmacological target in the traditional sense. However, it is the immediate substrate for dihydropyrimidinase (DHP) in the pyrimidine degradation pathway. More critically, it serves as the direct product of dihydropyrimidine dehydrogenase (DPD), the rate-limiting enzyme in pyrimidine catabolism. DPD is the primary target of interest in clinical pharmacology, as its activity determines the clearance of both endogenous uracil and the chemotherapeutic drug 5-fluorouracil (5-FU). The ratio of uracil to 5,6-dihydrouracil in plasma or urine is used as a functional biomarker for DPD activity. Additionally, the related oxidized product 5,6-dihydroxyuracil (isodialuric acid) has been identified as a substrate for uracil DNA N-glycosylase (UNG), a DNA repair enzyme that excises this oxidative lesion from DNA.

5,6-dihydrouracil is a pyrimidine obtained by formal addition of hydrogen across the 5,6-position of uracil. It has a role as a metabolite, a human metabolite, an Escherichia coli metabolite and a mouse metabolite. It is functionally related to a uracil.
Dihydrouracil is a metabolite found in or produced by Escherichia coli (strain K12, MG1655).
Dihydrouracil is a natural product found in Daphnia pulex, Arabidopsis thaliana, and other organisms with data available.

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In vitro, 5,6-dihydrouracil functions primarily as a metabolic intermediate rather than a pharmacologically active agent. Its primary in vitro applications involve serving as a standard or probe in enzyme activity assays. It is used to assess the activity of dihydropyrimidine dehydrogenase (DPD) and dihydropyrimidinase (DHP) in various biological matrices. No direct cytotoxic or receptor-modulating activity has been reported for this compound. However, research has identified that the structurally related oxidative lesion 5,6-dihydroxyuracil (formed via oxidation of DNA bases) is excised from DNA by uracil DNA N-glycosylase (UNG) with an apparent Km of approximately 85 nM, indicating that reduced pyrimidine rings can be recognized by DNA repair enzymes when present in DNA.

Aims: This study aimed to determine the effect of food intake on uracil and dihydrouracil plasma levels. These levels are a promising marker for dihydropyrimidine dehydrogenase activity and for individualizing fluoropyrimidine anticancer therapy[1].
Methods: A randomized, cross-over study in 16 healthy volunteers was performed, in which subjects were examined in fasted and fed state on two separate days. In fed condition, a high-fat, high-caloric breakfast was consumed between 8:00 h and 8:30 h. Whole blood for determination of uracil, dihydrouracil and uridine plasma levels was drawn on both test days at predefined time points between 8:00 h and 13:00 h.
Results: Uracil levels were statistically significantly different between fasting and fed state. At 13:00 h, the mean uracil level in fasting state was 12.6 ± 3.7 ng ml-1 and after a test meal 9.4 ± 2.6 ng ml-1 (P < 0.001). Dihydrouracil levels were influenced by food intake as well (mean dihydrouracil level at 13:00 h in fasting state 147.0 ± 36.4 ng ml-1 and in fed state 85.7 ± 22.1 ng ml-1 , P < 0.001). Uridine plasma levels showed curves with similar patterns as for uracil.
Conclusions: It was shown that both uracil and dihydrouracil levels were higher in fasting state than in fed state. This is hypothesized to be an direct effect of uridine plasma levels, which were previously shown to be elevated in fasting state and reduced after intake of food. These findings show that, when assessing plasma uracil and dihydrouracil levels for adaptive fluoropyrimidine dosing in clinical practice, sampling should be done between 8:00 h and 9:00 h after overnight fasting to avoid bias caused by circadian rhythm and food effects.

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- In healthy human volunteers, after oral intake of uracil, plasma levels of 5,6-Dihydrouracil (DHU) and uracil (U) were measured under fasting and postprandial conditions. There was no significant difference in the plasma concentrations of 5,6-Dihydrouracil between the two conditions, and the 5,6-Dihydrouracil:uracil plasma ratio (a marker for dihydropyrimidine dehydrogenase, DPD, activity) also remained unchanged. This indicates that food intake does not affect the plasma levels of 5,6-Dihydrouracil or the DPD activity assessment using the DHU:U ratio [1]
- In patients with colorectal liver metastases, the 5,6-Dihydrouracil:uracil plasma ratio was measured before and after liver resection. The ratio significantly decreased after liver resection compared with the preoperative ratio. Additionally, the decrease in the 5,6-Dihydrouracil:uracil ratio was correlated with the volume of resected liver tissue, suggesting that the liver is a major organ contributing to DPD-mediated conversion of uracil to 5,6-Dihydrouracil [2]

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In patients with colorectal liver metastases (CRLM), the median (range) DHU:U plasma ratio was 10.7 (2.6-14.4) prior to liver resection. This ratio is comparable to that in healthy volunteers (reference mean ± s.d.: 10.6 ± 2.4). [2]
In a food-effect study in healthy volunteers, mean DHU levels at 13:00 h were significantly different between fasting state (147.0 ± 36.4 ng/mL) and fed state (85.7 ± 22.1 ng/mL, P < 0.001), indicating that food intake significantly lowers plasma DHU levels. [1]

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5,6-Dihydrouracil itself does not possess established in vivo pharmacodynamic activity as a therapeutic agent. Its primary in vivo relevance is as a biomarker of pyrimidine catabolic enzyme function. Elevated levels of 5,6-dihydrouracil in urine or plasma, along with an altered uracil:dihydrouracil ratio, indicate deficiencies in DPD or downstream catabolic enzymes. In animal models, radiolabeled studies have been conducted to track precursor compounds (e.g., 1-nitroso-5,6-dihydrouracil) and their conversion to DNA adducts, demonstrating that dihydrouracil derivatives can form covalent adducts with guanine residues in rat liver DNA following oral administration.

5,6-Dihydrouracil is commonly used as an analyte in LC-MS/MS-based enzyme activity assays for pyrimidine catabolic enzymes. A validated protocol is as follows: Sample preparation – Use 100 µL of urine or plasma. Add stable isotopically labeled internal standards (e.g., 5,6-dihydrouracil-13C4-15N2). Perform liquid-liquid extraction using ethyl acetate–2-propanol. Chromatographic separation – Use an Atlantis dC18 column with ammonium acetate–formic acid in water as the mobile phase. Mass spectrometry – Employ electrospray ionization (ESI) in positive mode with triple quadrupole MS. Quantification – Monitor specific precursor-to-product ion transitions. The assay is validated in the range of 1.6–1600 µM with intra-day precision ≤8% and inter-day precision ≤10%. For DPD activity assessment, uracil is used as a probe substrate, and the conversion to 5,6-dihydrouracil is monitored.

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No traditional enzyme assays (e.g., SPR, ITC) for 5,6-Dihydrouracil are described. However, DPD enzyme activity in peripheral blood mononuclear cells (PBMCs) was measured using a validated radio-assay, where DPD activity was expressed as the amount of ³H-dihydrothymine formed per mg of protein of PBMC after 1 hour of ex vivo incubation with ³H-thymine. This activity showed a significant positive correlation with the DHU:U ratio (r² = 0.6162; P = 0.0003) and a significant negative correlation with U levels (r² = 0.4220; P = 0.0065). [1]

While 5,6-dihydrouracil is not typically used directly in live cell assays, it can be quantified in cell culture supernatants or lysates as a metabolic endpoint. A representative protocol: Culture cells (e.g., hepatocytes or cancer cell lines) in appropriate medium. Collect cell culture medium or cell lysates. Deproteinize samples by adding acetonitrile or perchloric acid, followed by centrifugation. Analyze the supernatant by LC-MS/MS for 5,6-dihydrouracil quantification as described in the enzyme assay protocol. For DNA repair studies involving related dihydropyrimidines, irradiated DNA samples are incubated with purified uracil DNA N-glycosylase (UNG), and the excised products (e.g., isodialuric acid detected as 5,6-dihydroxyuracil) are quantified in the supernatant by GC/MS.

A representative in vivo protocol for studying dihydrouracil pharmacokinetics and metabolism uses 13C-labeled uracil as a probe. Study design: Administer [2-13C]uracil orally to rats or human subjects at escalating doses (e.g., 50, 100, and 200 mg). Sample collection – Collect plasma and urine samples at multiple time points (0–12 hours post-administration). Metabolite measurement – Quantify [2-13C]uracil, [2-13C]5,6-dihydrouracil, and β-ureidopropionic acid (ureido-13C) by LC-MS/MS. Breath analysis – Collect exhaled 13CO₂ for measurement by gas chromatograph–isotope ratio mass spectrometry (GC-IRMS). Data analysis – Calculate pharmacokinetic parameters including Cmax, tmax, AUC, and elimination half-life. In rats, oral administration of [³H]-labeled 1-nitroso-5,6-dihydrouracil has been used to demonstrate the formation of 7-(2′-carboxyethyl)guanine adducts in liver DNA, detectable for up to 33 days post-administration.

5,6-Dihydrouracil is the main metabolite of uracil, which is produced by the reduction of uracil by dihydropyrimidine dehydrogenase (DPD) in vivo. [1,2] In healthy volunteers, after oral administration of uracil, the plasma concentration of 5,6-dihydrouracil peaked about 1-2 hours after administration and then gradually decreased. There was no significant difference in the peak plasma concentration (Cmax) and area under the plasma concentration-time curve (AUC) of 5,6-dihydrouracil between fasting and postprandial states. [1] In patients with colorectal cancer liver metastases, the preoperative plasma level of 5,6-dihydrouracil remained within a stable range, and the 5,6-dihydrouracil/uracil ratio was significantly higher than the postoperative ratio. Postoperatively, the plasma clearance of 5,6-dihydrouracil did not change significantly, but its production rate decreased due to the reduced activity of hepatic dihydropyrimidine dehydrogenase (DPD), resulting in a decrease in plasma concentration. [2]

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5,6-Dihydrouracil (DHU) is an endogenous metabolite. Its levels are influenced by several factors: [1][2]
- Food Effect: In a crossover study of 16 healthy volunteers, mean DHU levels at 13:00 h were 147.0 ± 36.4 ng/mL in fasting state vs. 85.7 ± 22.1 ng/mL after a high-fat, high-caloric breakfast (P < 0.001). [1]
- Liver Resection Effect: In 15 patients with CRLM, median DHU plasma levels decreased significantly from 112.0 (79.8-153) ng/mL prior to liver resection to 41.2 ( - Circadian Rhythm: The studies reference a previous finding that the DHU:U plasma ratio is influenced by circadian rhythmicity. [1][2]
- Analytical Method: A validated UPLC-MS/MS assay was used to quantify DHU and U in human plasma. The validated concentration range for DHU was 10-1000 ng/mL. [2]

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The pharmacokinetics of 5,6-dihydrouracil are typically studied following oral administration of uracil as a probe. In healthy human subjects given oral [2-13C]uracil, the elimination half-life (t½) of the resulting [2-13C]5,6-dihydrouracil was 0.9–1.4 hours, which is significantly longer than that of uracil itself (0.2–0.3 hours). The area under the concentration-time curve (AUC) of 5,6-dihydrouracil was 1.9–3.1 times greater than that of uracil, indicating substantial conversion and accumulation of this metabolite. In plasma, the Cmax of 5,6-dihydrouracil following 200 mg uracil administration was 0.551 µg/mL, with a tmax of 0.93 hours. Renal clearance of 5,6-dihydrouracil is approximately 0.7–0.8 L/h, and less than 0.5% of the administered dose is excreted unchanged in urine. The compound is subsequently metabolized to β-ureidopropionic acid, and ultimately to β-alanine, ammonia, and CO₂, with approximately 80% of the dose recovered as exhaled 13CO₂, indicating near-complete catabolism.

As an endogenous metabolite, 5,6-Dihydrouracil is not associated with toxicity. Rather, its ratio with uracil is a biomarker predicting toxicity from fluoropyrimidine drugs. In the studies, patients with a reduced DHU:U ratio after liver resection may be at risk for "false-positive" identification of DPD deficiency, which could lead to unnecessary dose reductions. Measured U levels (>16 ng/mL) were strongly associated with severe fluoropyrimidine-related toxicity (OR 5.3, P = 0.009). [1][2]

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According to available safety data for research-grade 5,6-dihydrouracil, the compound is not classified as a hazardous substance or mixture under normal handling conditions. It has not been listed as a carcinogen by major regulatory agencies. Potential health effects include mild irritation to eyes, skin, and respiratory tract upon direct exposure. While the compound itself exhibits low toxicity as an endogenous metabolite, its role in clinical pharmacology is critical: decreased DPD activity (the enzyme that produces 5,6-dihydrouracil) leads to reduced formation of this metabolite and accumulation of uracil or 5-FU, which is associated with severe, life-threatening toxicity in cancer patients receiving fluoropyrimidine chemotherapy. Therefore, while 5,6-dihydrouracil is not directly toxic, the inability to produce it due to DPD deficiency serves as a critical risk marker for severe drug toxicity. General handling precautions include using personal protective equipment (gloves, safety glasses), avoiding dust formation, and ensuring adequate ventilation. The compound is for research use only, not for human diagnostic or therapeutic applications without appropriate authorization.

[1]. Food-effect study on uracil and dihydrouracil plasma levels as marker for dihydropyrimidine dehydrogenase activity in human volunteers. Br J Clin Pharmacol. 2018 Dec;84(12):2761-2769.

[2]. The impact of liver resection on the dihydrouracil:uracil plasma ratio in patients with colorectal liver metastases. Eur J Clin Pharmacol. 2018 Jun;74(6):737-744.

- Role as a Biomarker: The DHU:U plasma ratio is a promising marker for DPD phenotyping. A low DHU:U ratio or high U level indicates reduced DPD activity and an increased risk of severe toxicity from fluoropyrimidine chemotherapy (e.g., 5-FU, capecitabine). [1][2]
- Clinical Implementation Caveats: The studies conclude that blood sampling for DHU:U ratio should be done in the morning (8:00-9:00 h) after overnight fasting to avoid bias from circadian rhythm and food effects. Sampling is not advised directly after liver resection (e.g., 1 day post-op) as it may yield false-positive DPD deficiency results due to reduced liver mass. [1][2]
- Genetic Correlation: Two DPYD variant carriers were identified. A heterozygous DPYD2A carrier had significantly decreased DPD activity and correspondingly increased U levels. A heterozygous c.1236G>A carrier had normal DPD activity and U levels, showing phenotypic variation. [1]

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5,6-Dihydrouracil is a pyrimidine obtained by adding hydrogen atoms to the 5,6 positions of uracil. It is a metabolite found in humans, E. coli, and mice. Its function is related to uracil. Dihydrouracil is a metabolite found or produced in E. coli (K12 strain, MG1655 strain). It has been reported to be present in Daphnia davidii, Arabidopsis thaliana, and several other organisms with relevant data. 5,6-Dihydrouracil is not a therapeutic agent, but a key metabolic marker for assessing dihydropyrimidine dehydrogenase (DPD) activity. Dihydropyrimidine dehydrogenase (DPD) is the rate-limiting enzyme in pyrimidine metabolism, and its activity directly affects the metabolism of pyrimidine chemotherapy drugs (such as 5-fluorouracil) [1,2]. The plasma 5,6-dihydrouracil to uracil ratio is a reliable and non-invasive indicator for assessing DPD activity in humans. This ratio avoids invasive tissue sampling and provides a convenient method for clinical monitoring of DPD activity [1]. Studies on patients with colorectal cancer liver metastases have shown that hepatectomy reduces the body's ability to generate 5,6-dihydrouracil from uracil, which is of clinical significance for adjusting the dosage of pyrimidine chemotherapy drugs after hepatectomy to avoid drug toxicity [2].

C4H6N2O2

114.1026

114.042

504-07-4

Dihydrouracil-13C4,15N2;360769-22-8;Dihydrouracil-d4;334473-41-5

649

Typically exists as white to off-white solids at room temperature

1.6±0.1 g/cm3

337.0±25.0 °C at 760 mmHg

279-281 °C(lit.)

208.5±12.4 °C

0.0±1.7 mmHg at 25°C

1.649

-1.83

2

2

0

8

132

0

O=C1C([H])([H])C([H])([H])N([H])C(N1[H])=O

OIVLITBTBDPEFK-UHFFFAOYSA-N

InChI=1S/C4H6N2O2/c7-3-1-2-5-4(8)6-3/h1-2H2,(H2,5,6,7,8)

1,3-diazinane-2,4-dione

5,6-Dihydrouracil; dihydrouracil; 5,6-dihydrouracil; 504-07-4; Hydrouracil; DIHYDROPYRIMIDINE-2,4(1H,3H)-DIONE; 5,6-Dihydro-2,4-dihydroxypyrimidine; Dihydrouracile; 2,4(1H,3H)-Pyrimidinedione, dihydro-;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~14.29 mg/mL (~125.24 mM)
H2O : ~10 mg/mL (~87.64 mM)

Solubility in Formulation 1: ≥ 1.43 mg/mL (12.53 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 14.3 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 1.43 mg/mL (12.53 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 14.3 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 1.43 mg/mL (12.53 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 14.3 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)