Endogenous Metabolite
Uracil is a natural pyrimidine nucleobase found in RNA. In itself, it is not a drug with a specific protein target like an enzyme or receptor. Its derivatives and analogs are designed to target various biological processes. This review discusses many uracil derivatives with different targets, but specific target information (e.g., IC50, Ki) for the parent compound uracil is not provided. The derivatives mentioned target enzymes such as thymidine phosphorylase (TP), glycogen phosphorylase (GP), Plasmodium falciparum dUTPase (PfdUTPase), HIV-1 reverse transcriptase, and others. [1]
Uracil itself does not directly bind to specific therapeutic targets in a drug-like manner; rather, it functions as a natural metabolite and a building block for RNA synthesis. However, uracil derivatives exert their effects through various mechanisms, including inhibition of thymidylate synthase, viral DNA polymerases, dipeptidyl peptidase-4 (DPP-4), and modulation of P2 receptors. In the clinic, uracil is utilized as a biochemical modulator in combination chemotherapy (e.g., UFT regimen: uracil + ftorafur), where it competitively inhibits dihydropyrimidine dehydrogenase (DPD), thereby prolonging the half-life and enhancing the bioavailability of 5-fluorouracil (5-FU).

Uracil itself exhibits limited direct biological activity as a single agent. However, its derivatives demonstrate significant in vitro pharmacological effects. For instance, 3-oxauracil (a uracil analogue) showed significant cytotoxic activity against various human tumor cell lines, including pancreatic (RWP-2, MiaPaCa-2, PANC-1), colon (HT-29), neuroendocrine (COLO 320DM), and lung cancer (SK-MES-1) cells at a concentration of 103 μM. Additionally, uracil-based compounds have demonstrated in vitro inhibitory activity against viral infections such as H1N1 influenza virus (EC50 as low as 0.3 μM), coxsackievirus B4, and antibacterial/antifungal properties. Uracil derivatives have also been shown to inhibit pathogens like Trypanosoma brucei and Leishmania mexicana in cell cultures.

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This review article is an effort to summarize recent developments in researches providing uracil derivatives with promising biological potential. This article also aims to discuss potential future directions on the development of more potent and specific uracil analogues for various biological targets. Uracils are considered as privileged structures in drug discovery with a wide array of biological activities and synthetic accessibility. Antiviral and anti-tumour are the two most widely reported activities of uracil analogues however they also possess herbicidal, insecticidal and bactericidal activities. Their antiviral potential is based on the inhibition of key step in viral replication pathway resulting in potent activities against HIV, hepatitis B and C, the herpes viruses etc. Uracil derivatives such as 5-fluorouracil or 5-chlorouracil were the first pharmacological active derivatives to be generated. Poor selectivity limits its therapeutic application, resulting in high incidences of gastrointestinal tract or central nervous toxicity. Numerous modifications of uracil structure have been performed to tackle these problems resulting in the development of derivatives exhibiting better pharmacological and pharmacokinetic properties including increased bioactivity, selectivity, metabolic stability, absorption and lower toxicity. Researches of new uracils and fused uracil derivatives as bioactive agents are related with modifications of substituents at N(1), N(3), C(5) and C(6) positions of pyrimidine ring. This review is an endeavour to highlight the progress in the chemistry and biological activity of the uracils, predominately after the year 2000. In particular are presented synthetic methods and biological study for such analogues as: 5-fluorouracil or 5-chlorouracil derivatives, tegafur analogues, arabinopyranonucleosides of uracil, glucopyranonucleosides of uracil, liposidomycins, caprazamycins or tunicamycins, tritylated uridine analogues, nitro or cyano derivatives of uracil, uracil-quinazolinone, uracil-indole or uracil-isatin-conjugates, pyrimidinophanes containing one or two uracil units and nitrogen atoms in bridging polymethylene chains etc. In this review is also discussed synthesis and biological activity of fused uracils having uracil ring annulated with other heterocyclic ring[1].

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The review focuses on the activities of its modified analogs. For example, 5-fluorouracil (5-FU) inhibits RNA replication enzymes. Many synthesized uracil derivatives show anticancer, antiviral, antibacterial, and antiparasitic activities in cell-based assays. [1]
A study on the excision of uracil and its halogenated derivatives (5-fluorouracil/FU, 5-chlorouracil/CIU, 5-bromouracil/BrU) from DNA by human thymine DNA glycosylase (hTDG) was mentioned. The activity (kmax) for excising these bases from GX substrates depended significantly on the 5' flanking base pair. For instance, the kmax for excising CIU decreased by 6-fold, 11-fold, and 82-fold for TpGCIU, GpGCIU, and ApGCIU contexts, respectively, compared to CpGCIU. The activity for excising G FU, GCIU, and GBrU with any 5' flanking pair met or exceeded the activity for CpG T excision. [1]

In vivo studies in mice showed that uracil derivatives exhibit central nervous system effects, with uracil and thymine increasing spontaneous activity at lower doses and decreasing it at higher doses. As a component of UFT (uracil + ftorafur), the combination demonstrated clinical efficacy in cancer patients. A phase I study reported that in previously untreated patients with advanced colorectal cancer, two of eight patients achieved partial responses with UFT (350 mg/m²) plus leucovorin. Limited in vivo studies in mice demonstrated that 3-oxauracil (3.23 mg/kg, i.p. daily for two weeks) exhibited antiviral effects without significant toxicity.

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In vivo data are mentioned for some derivatives, such as a gold(I) complex of a uracil derivative (6-amino-1-methyl-5-nitrosouracil, MANU) which decreased experimental glioma tumor growth to about one-tenth after seven days of treatment in an animal model. [1]

Uracil is involved in DPD (dihydropyrimidine dehydrogenase) enzyme activity assays as a substrate to assess metabolic capacity. A standard protocol for a uracil loading test: Uracil is administered orally at a dose of 500 mg/m². Blood samples (2-4 mL) are collected into lithium heparin tubes at multiple time points (e.g., t = 15, 30, 45, 60, 80, 100, 120, 150, 180, and 220-240 minutes) following uracil intake. Plasma levels of uracil and its metabolite dihydrouracil (DHU) are quantified by HPLC-UV. DPD activity is measured in peripheral blood mononuclear cells (PBMCs). The U/DHU ratio at specific time points is calculated to assess systemic DPD activity, with higher ratios indicating DPD deficiency.

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A modified thymidine phosphorylase (TP) bioassay is described for evaluating uracil derivatives. The bioassay used recombinant Escherichia coli TP, expressed in E. coli, as the enzyme and thymidine as the substrate. The inhibitory activity of synthesized compounds was evaluated using this assay. For example, a 5-chlorouracil-linked-pyrazolo[1,5-a][1,3,5]triazine derivative (compound 262, R = 4-F5S-phenyl) showed an IC50 of 0.04 µM, which was around 800 times more potent than the lead compound 7-deazaxanthine (7DX, IC50 = 32 µM) under the same conditions. This compound was found to be a non-competitive inhibitor. [1]
An assay for evaluating inhibitors of Plasmodium falciparum and Leishmania major dUTPase enzymes, as well as the human enzyme, is mentioned. Compounds were tested against recombinant enzymes to measure selectivity. For instance, a tritylated deoxyuridine analogue (5'-tritylamino-2',5'-dideoxyuridine 124, X=OH) showed a Ki of 0.2 µM against P. falciparum dUTPase with over 200-fold selectivity compared to the human enzyme. [1]
A kinetic experiment for evaluating uracil derivatives as inhibitors of glycogen phosphorylase (GP) is mentioned. The best inhibitor identified was 1-(β-D-glucopyranosyl)-5-ethynyluracil (99) with a Ki of 4.7 µM. [1]

A typical cell proliferation inhibition assay for evaluating uracil derivative cytotoxicity: Human tumor cell lines (e.g., HT-29 colon cancer cells) are seeded in appropriate culture medium at densities of approximately 5 × 10³ - 1 × 10⁴ cells per well in 96-well plates. Following overnight attachment, cells are treated with varying concentrations of the test compound (e.g., uracil or its derivatives) dissolved in DMSO and diluted in culture medium, typically ranging from 0.1 - 200 μM. After 48-72 hours of incubation at 37°C in 5% CO₂, cell viability is assessed using MTT, XTT, or [³H]-thymidine incorporation assays. Untreated controls and vehicle controls (DMSO) are included for comparison.

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The sulforhodamine B (SRB) assay is mentioned for evaluating cytotoxic activity. Compounds were tested against human cancer cell lines such as cervical (HeLa), oral (KB), and breast (MCF-7). For example, a phosphoramidate derivative of 3'-azido-2',3'-dideoxy-5-fluorouridine (25, R = CH2CH3) displayed the highest activity in all investigated cancer cells, much higher than the parent nucleoside. [1]
The MTT assay is mentioned for cytotoxic evaluation against human cancer cell lines (e.g., HeLa, MCF-7, DU145). For instance, uracil-isatin conjugates (107) were evaluated, with some showing IC50 values as low as 13.90 µM against DU145 cells. [1]
Antiviral activity assays are described. Compounds were tested against viruses such as herpes simplex virus type 1 (HSV-1), varicella-zoster virus (VZV), human cytomegalovirus (HCMV), dengue virus (DENV), and yellow fever virus (YFV) in appropriate cell lines (e.g., CCL-81 for HSV, AD-169 for HCMV, MDCK for influenza). For example, 5-(thien-2-yl)-2'-deoxyuridines (40) exhibited marked activity against HSV-1, and some halogenated analogs (41) were equipotent to brivudin (BVDU). A fluorocyclopropyl uracil nucleoside (55) showed moderate anti-HCMV activity (10.61 µg/mL in AD-169 strain). [1]
Cytotoxicity (CC50) was measured on normal human intestinal cell line (H4) and tumor cell lines (Caco-2, melanoma, MCF-7) for some unsaturated uracil nucleosides. Compound 65 was found to be potent in the MCF-7 breast carcinoma cell line. [1]
For uracil-derived pyrimidinophanes, antibacterial and antifungal activity was investigated against Gram-negative bacteria (Pseudomonas aeruginosa, Escherichia coli), Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis, Enterococcus faecalis), pathogenic fungi (Aspergillus niger, Trichophyton mentagrophytes, Aspergillus fumigatus), and yeast (Candida albicans). Activity increased with polymethylene chain length and upon introduction of an n-decyl substituent. [1]

For assessing central nervous system effects of pyrimidines including uracil: Adult male C-57 mice are administered the test compound via intraperitoneal (i.p.) or parenteral routes. Spontaneous activity is measured using activity cages or similar monitoring systems over defined time periods (e.g., 20-60 minutes post-administration). Lower doses (e.g., 10-50 mg/kg) are tested for stimulant effects, while higher doses (e.g., 50-200 mg/kg) are tested for depressant effects. Results are expressed as percentage change from baseline activity, and ED50 values for stimulation and depression are calculated.

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The review describes an in vivo study for a gold(I) complex of a uracil derivative (6-amino-1-methyl-5-nitrosouracil, MANU) in an animal model of experimental glioma. The result mentioned was that after seven days of treatment, the gold compound decreased tumor growth to about one-tenth compared to vehicle-treated animals. [1]

In humans, following oral administration of uracil (500 mg/m²) in healthy volunteers, pharmacokinetic parameters are as follows: AUCuracil (T=180) = 865.1 ± 240.3 mg·min/L, Cmax uracil = 14.4 ± 4.7 mg/L, and AUCdihydrouracil (T=220) = 439.3 ± 134.5 mg·min/L. The uracil/dihydrouracil (U/DHU) ratio decreases over time, from approximately 0.9 at 100 minutes to 0.1 at 180 minutes in individuals with normal DPD activity. In DPD-deficient patients, the U/DHU ratio is significantly higher at all time points, demonstrating reduced metabolic conversion of uracil to dihydrouracil. Uracil is rapidly absorbed and metabolized primarily by DPD in the liver and peripheral tissues.

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The review points out that 5-fluorouracil (5-FU, a uracil derivative) has poor selectivity, short half-life, and wide distribution, which limits its therapeutic application. However, the review does not provide specific ADME/PK parameters (absorption, distribution, metabolism, excretion, half-life, bioavailability) of the parent compound uracil. [1]
For some triphenylmethylated uracil analogs developed as dUTPase inhibitors of Plasmodium falciparum, preliminary ADME studies have shown that some lead compounds have drug activity. However, the review also does not provide specific parameters. [1]

Uracil itself is generally considered safe at physiological concentrations as an endogenous metabolite. However, in the context of combination chemotherapy (UFT), the primary dose-limiting toxicities are gastrointestinal, including diarrhea, nausea, and vomiting, with dose-limiting toxicity observed at 350 mg/m² UFT (uracil + ftorafur) in previously-chemotherapy-treated patients. Mild fatigue and transient hyperbilirubinemia are also commonly reported. At the molecular level, DPD-deficient patients are at significantly increased risk for severe toxicity when exposed to 5-FU or uracil-containing prodrugs, as the genetic deficiency leads to accumulation of toxic metabolites. As a neat chemical, uracil is listed with RTECS number YQ8650000; general handling precautions include avoiding contact with strong oxidizing agents.

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The review points out that 5-fluorouracil (5-FU) has side effects such as gastrointestinal or central nervous system toxicity due to its poor selectivity. [1]
For a series of uracil-benzodioxane hybrids (cyclic and acyclic 5-FU O,N-acetal 150, 151), the IC50 values of all compounds against MCF-7 cells were in the micromolar range. One of the compounds (151, R1=NO2, R2=H) was the most cytotoxic. Another compound (151, R1=R2=H) induced apoptosis and G0/G1 cell cycle arrest in MCF-7 cells. [1]
In the study of 5-[1-(2-haloethyl (or nitro)ethoxy-2-iodoethyl)]-2'-deoxyuridine (46), all compounds showed low host cell toxicity. [1]
Serum parameter analysis of the MANU gold(I) complex indicated fewer adverse reactions after treatment. [1]
Structure-activity relationship analysis of 5,6-substituted 1-[(2-hydroxyethoxy)methyl]uracil showed that cytotoxicity depended on lipophilicity and steric hindrance parameters. [1]

[1]. In search of uracil derivatives as bioactive agents. Uracils and fused uracils: Synthesis, biological activity and applications. Eur J Med Chem. 2015 Jun 5;97:582-611.

Uracil is a common naturally occurring pyrimidine nucleobase, with its pyrimidine ring substituted at positions 2 and 4 by two oxo groups. It is present in RNA, pairs with adenine, and substitutes for thymine during DNA transcription. Uracil has multiple functions, including as a prodrug, a human metabolite, a Daphnia magna metabolite, a Saccharomyces cerevisiae metabolite, an Escherichia coli metabolite, a mouse metabolite, and an allergen. It is both a pyrimidine nucleobase and a pyrimidinone. It is a tautomer of (4S)-4-hydroxy-3,4-dihydropyrimidin-2(1H)-one. Uracil is a metabolite found or produced in Escherichia coli (K12 strain, MG1655 strain). Uracil has been reported in Hamigela avilania, Micrococcus microcarpa, and other organisms with relevant data.
Uracil is a metabolite found or produced in Saccharomyces cerevisiae.
It is one of the four nucleotide bases in RNA.
See also: Pyrimidine (subclass).

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Background: Uracil is a common natural pyrimidine derivative and one of the four nucleotide bases in RNA that binds to adenine. In DNA, it is replaced by thymine. It can be considered a demethylated form of thymine. Uracil undergoes amide-imine (lactam-lactam) tautomerism, with the lactam form being predominant at pH 7. Uracil is a weak acid. [1]
Preferred structure: Uracil is considered a preferred structure in drug discovery due to its broad bioactivity, ease of synthesis, and the ability to impart drug-like properties upon modification at N1, N3, C5, and C6 sites. [1]
Derivative activity: Uracil analogs are most commonly reported to have antiviral and antitumor activities, but also herbicidal, insecticidal, and bactericidal activities. [1]
Mechanism analysis (e.g., 5-fluorouracil): 5-fluorouracil (5-FU) is an anti-metabolic pyrimidine analog. Due to its similar structure to uracil but different chemical properties, it can inhibit RNA replicase, thereby blocking RNA synthesis and inhibiting cancer cell growth. [1]
Metabolic transformation: This review describes the specific chemical transformation of uracil derivatives (β-hydroxy-N-methylvaline/βHOMeVal in basidiomycin A) under alkaline conditions, involving reverse aldol condensation or dehydration reactions, but this is not the general metabolic pathway of uracil itself. [1]

C4H4N2O2

112.0868

112.027

C, 42.86; H, 3.60; N, 24.99; O, 28.55

66-22-8

66-22-8 (uracil); 3083-77-0 [1-beta-D-Arabinofuranosyluracil (Uracil 1-β-D-arabinofuranoside)]; 462-88-4 (Ureidopropionic acid); 504-07-4 (5,6-Dihydrouracil); 66-75-1 (Uramustine, Uracil mustard); 141-90-2 (2-Thiouracil); 58-96-8 (Uridin; β-Uridine)

1174

Typically exists as white to light yellow solids at room temperature

1.5±0.1 g/cm3

440.5±37.0 °C at 760 mmHg

330°C

220.2±26.5 °C

0.0±1.1 mmHg at 25°C

1.640

-2.55

2

2

0

8

161

0

O=C1N([H])C([H])=C([H])C(N1[H])=O

ISAKRJDGNUQOIC-UHFFFAOYSA-N

InChI=1S/C4H4N2O2/c7-3-1-2-5-4(8)6-3/h1-2H,(H2,5,6,7,8)

Pyrimidine-2,4(1H,3H)-dione

2,4-Dioxopyrimidine; 2,4-Pyrimidinedione; Pirod; uracil; 66-22-8; 2,4-Dihydroxypyrimidine; 2,4(1H,3H)-Pyrimidinedione; pyrimidine-2,4(1H,3H)-dione; pyrimidine-2,4-diol; Pyrod; 2,4-Pyrimidinediol; Pyrod.

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 : ≥ 25 mg/mL (~223.04 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (22.30 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 25.0 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: ≥ 2.5 mg/mL (22.30 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 25.0 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: ≥ 2.5 mg/mL (22.30 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 25.0 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.)