Selective inhibitor of neuronal nitric oxide synthase (nNOS). Competitive inhibition with a Ki of 20 μM. Shows low activity against inducible NOS (iNOS, <25% inhibition at 500 μM) and endothelial NOS (eNOS, <15% inhibition at 500 μM). [1]

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2-Thiouracil does not target a specific enzyme or receptor. Its mechanism of action involves nucleophilic addition to quinonoid intermediates in the melanin biosynthesis pathway, specifically: dopaquinone (forming the 6-S-thiouracil-DOPA adduct), 5,6-indolequinone from DHI (forming C-2 and C-3 substituted adducts), and the o-indolequinone from DHICA (forming a C-4 substituted adduct). The provided document does not include IC50, Ki, or EC50 values for these interactions. [2]
2-Thiouracil exerts its effects through multiple mechanisms. Its primary classical target is thyroid peroxidase (TPO), the enzyme responsible for iodination of thyroglobulin in thyroid hormone synthesis . By inhibiting TPO, the compound blocks the production of thyroxine (T4) and triiodothyronine (T3), thereby reducing thyroid hormone levels. In the context of melanoma targeting, 2-thiouracil acts as a substrate for tyrosinase, an enzyme involved in melanin biosynthesis. It undergoes nucleophilic attack on transient quinonoid intermediates in the melanin pathway—primarily dopaquinone and 5,6-indolequinones—leading to covalent incorporation into growing melanin polymers . Additionally, 2-thiouracil has been identified as a selective inhibitor of neuronal nitric oxide synthase (nNOS), antagonizing tetrahydrobiopterin-dependent enzyme activation and dimerization .

- Reaction with DOPA/Tyrosine: In the tyrosinase-catalyzed oxidation of DOPA (1.5 mM) in the presence of TU (3 mM, 2:1 molar excess), the reaction course was markedly altered. Besides some DHI, a complex mixture of products was formed. The major product was identified as 6-S-thiouracil-DOPA (TU-DOPA). Other peaks (III-VIII) corresponded to DHI-TU adducts. [2]
- Reaction with DHI: When DHI (1.5 mM) was oxidized by tyrosinase in the presence of TU (3 mM), the normal oligomerization pattern of DHI was virtually suppressed. Instead, new products were isolated and identified as: a 1:1 DHI-TU adduct substituted at the C-3 position (compound 1, 5,6-dihydroxy-3-[(4-hydroxypyrimidin-2-yl)thio]indole), a 1:1 adduct substituted at the C-2 position (compound 2), a symmetric dimer of the 1:1 adduct linked at the C-4 and C-4' positions (compound 3), and a trimer containing two TU units (compound 4). The overall isolated yield of products 1-4 was about 20%. [2]
- Reaction with DHICA: Oxidation of DHICA (1.5 mM) by tyrosinase in the presence of TU (3 mM) led to the formation of DHICA-TU conjugates. A 1:1 adduct substituted at the C-4 position (compound 5, 2-carboxy-5,6-dihydroxy-4-[(4-hydroxypyrimidin-2-yl)thio]indole) was isolated. Additionally, a dimer containing one TU unit (compound 6) and a trimer bearing one TU unit (compound 7) were characterized. [2]
- Reactivity of TU-Indole Adducts: Upon enzymatic oxidation with tyrosinase, adducts 1, 2, and 5 smoothly decomposed to afford grayish materials with featureless absorption in the 400-600 nm range, without significant scattering due to insoluble melanin-like pigments, indicating poor melanogenic capacity. [2]

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Incubation of recombinant rat nNOS with TU resulted in a concentration-dependent inhibition of enzyme activity, with an IC50 of 50 ± 5 μM, as determined by the L-[14C]citrulline formation assay. [1]
At 1 mM concentration, TU caused a 51 ± 2% inhibition of NO formation (measured via nitrate/nitrite accumulation) by recombinant nNOS under the assay conditions. [1]
At 100 μM concentration, TU caused approximately 60% inhibition of H2O2 production by nNOS in the absence of L-arginine and tetrahydrobiopterin (BH4). This inhibition is specific to the oxygenase domain activity, as TU did not affect cytochrome c reductase activity of nNOS (a reductase domain function) at the same concentration. [1]
TU (500 μM) antagonized BH4-induced dimerization of nNOS, as demonstrated by low-temperature SDS-PAGE, reducing the amount of the 320 kDa dimer band formed in the presence of 1 μM BH4. This inhibitory effect on dimerization was suppressed when the BH4 concentration was raised to 10 μM. [1]

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In vitro studies have demonstrated that 2-thiouracil derivatives, particularly when coordinated as ruthenium(II) complexes, exhibit potent cytotoxic effects against cancer cell lines. Treatment of HepG2 hepatocellular carcinoma cells with Ru(II)-2-thiouracil complexes resulted in increased phosphatidylserine exposure, loss of mitochondrial transmembrane potential, PARP cleavage, DNA fragmentation, chromatin condensation, and cytoplasmic shrinkage . Mechanistically, these complexes suppress liver cancer stem cells by targeting NF-κB and Akt/mTOR signaling pathways . In melanogenesis studies, when incubated with DOPA in the presence of tyrosinase, 2-thiouracil profoundly modifies normal melanin biosynthesis, favoring formation of addition products including 6-S-thiouracil-DOPA and various DHI-TU adducts .

- Incorporation into Melanoma Melanin: After intraperitoneal injection of [2-¹⁴C]-labeled 2-Thiouracil into B16 melanoma-bearing mice, most of the incorporation occurred within 30 minutes. At 30 minutes post-injection, specific incorporation was 25,000 cpm/mg of melanin; at 90 minutes, it was 23,000 cpm/mg; and at 180 minutes, it was 23,000 cpm/mg. TU administration caused a substantial decrease in the A₃₅₀ absorbance of the extracted melanin (from 0.071 ± 0.005 in control to 0.043 ± 0.004 at 180 min), indicative of altered pigment structure. The yield of PTCA (pyrrole-2,3,5-tricarboxylic acid) from chemical degradation of the tumor melanin also decreased after TU injection (from 1125 ± 62 ng/mg in control to 589 ± 40 ng/mg at 180 min). [2]

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In vivo studies in melanoma-bearing mice showed that injection of radiolabeled 2-thiouracil (TU) results in rapid and selective incorporation of the drug into tumor pigment . The incorporation substantially decreased the absorbance at 350 nm (A350) and the yields of pyrrole-2,3,5-tricarboxylic acid (PTCA), a melanin marker. In a hepatocellular carcinoma xenograft model (C.B-17 SCID mice with HepG2 cells), Ru(II)-2-thiouracil complexes (2 or 4 mg/kg, intraperitoneal, once daily for 21 days) inhibited tumor growth, achieving 53.6-65.4% tumor inhibition depending on the specific complex and dose . Treated animals showed no significant changes in body weight or vital organs (liver, kidney, lung, heart), indicating tolerable systemic toxicity . Studies with melanotic melanocytes demonstrated that the more lipophilic propylthiouracil analogue exhibits greater cellular accumulation and toxicity compared to 2-thiouracil itself .

For the primary NOS activity assay, recombinant nNOS was incubated in a reaction mixture containing Tris-HCl buffer (pH 7), NADPH, calcium chloride, BH4, calmodulin, FAD, FMN, and L-[U-14C]arginine at 37°C for 15 minutes. The reaction was stopped with ice-cold HEPES/EDTA buffer. The mixture was then passed through a cation-exchange column to retain unreacted arginine. The formed [14C]citrulline was eluted with water and quantified by liquid scintillation counting to determine enzyme activity. [1]
NO formation was alternatively assessed by measuring its metabolites, nitrate and nitrite. Recombinant nNOS was incubated with reaction components including L-arginine, and the accumulated nitrate/nitrite in the medium was quantified using a commercial colorimetric test kit. [1]
H2O2 formation by nNOS was determined using the ferrous thiocyanate assay. [1]
Cytochrome c reductase activity of nNOS was measured spectrophotometrically by monitoring NADPH-dependent reduction of cytochrome c in the absence of calcium and calmodulin. [1]

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No classical enzyme assays (e.g., for inhibition constant, IC50) for 2-Thiouracil are described in this document. The experiments focus on the chemical interaction of TU with enzymatically generated quinonoid intermediates in the melanin biosynthesis pathway. Tyrosinase (mushroom, 3900 units/mg) was used to catalyze the oxidation of DOPA, DHI, and DHICA in the presence or absence of TU at pH 7.0. [2]

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To study the interaction of 2-thiouracil with melanin biosynthesis enzymes, a tyrosinase-catalyzed oxidation assay is used. The protocol involves incubating 2-thiouracil with DOPA (3,4-dihydroxyphenylalanine) or DHI (5,6-dihydroxyindole) in the presence of tyrosinase. Reaction mixtures are maintained at appropriate buffer conditions (typically pH 6.8-7.4) at 25-37°C. The progress of the reaction can be monitored by UV-Vis spectrophotometry, measuring changes in absorption at 350 nm (characteristic of thiouracil incorporation) . The resulting melanin products are chemically degraded to yield pyrrole markers (PDCA and PTCA), which are then quantified by HPLC to assess the degree of 2-thiouracil incorporation . For thyroid peroxidase inhibition studies, the enzyme activity can be assessed by measuring the iodination of thyroglobulin or guaiacol oxidation in the presence of varying concentrations of 2-thiouracil .

For cytotoxicity evaluation, cancer cell lines (e.g., HepG2 hepatocellular carcinoma cells) are seeded in 96-well plates at appropriate densities (typically 5 × 10³ cells/well) and cultured overnight. Cells are then treated with varying concentrations of 2-thiouracil or its complexes (e.g., 1-100 μM) for 24-72 hours . Cell viability is assessed using MTT or CCK-8 assays. Apoptosis detection can be performed using Annexin V/PI staining followed by flow cytometry. Additional endpoints include measurement of mitochondrial membrane potential (using JC-1 or tetramethylrhodamine ethyl ester) and assessment of PARP cleavage by Western blot . For melanocyte accumulation studies, melanotic and amelanotic melanocytes are incubated with 2-thiouracil at concentrations ranging from 40 ng to 40 μg per 10⁶ cells, followed by analysis of intracellular drug concentrations using GC-MS .

- Animals: Pathogen-free female C57Bl/6 mice (6 weeks old) were used. [2]
- Tumor Model: Mice were challenged subcutaneously with B16 melanoma cells. Two weeks after challenge, when tumors were established, the mice were treated intravenously with 10 μCi of [2-¹⁴C]-labeled 2-Thiouracil. [2]
- Sample Collection: At times noted (30, 90, and 180 minutes), animals were euthanized by CO₂ inhalation. Tumors were dissected free of surrounding tissue, weighed, and rapidly frozen. [2]
- Melanin Extraction and Analysis: Tumors were homogenized in 1% acetic acid-ethanol (30:70, v/v) and centrifuged. Melanosomes were purified by sucrose density gradient ultracentrifugation. Dried melanin aliquots were dissolved in Soluene 350 and counted in a liquid scintillation counter. Spectrophotometric determination of melanin content was performed by the method of Ito et al. (1993). [2]

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A representative in vivo protocol for studying 2-thiouracil's anti-tumor activity uses murine xenograft models. Female C.B-17 SCID mice are subcutaneously injected with HepG2 cells (approximately 5 × 10⁶ cells in 0.1 mL PBS) to establish tumors. When tumors reach approximately 100-150 mm³, animals are randomized into treatment groups (n=5-8 per group). 2-Thiouracil derivatives (e.g., Ru(II) complexes) are administered intraperitoneally at doses of 2 or 4 mg/kg once daily for 21 consecutive days . Doxorubicin (2 mg/kg) serves as a positive control, and vehicle alone as negative control. Tumor dimensions are measured every 3-4 days using calipers, and tumor volume is calculated using the formula: volume = (length × width²)/2. At study termination, tumors are excised, weighed, and processed for histopathological examination. Body weights are monitored throughout the study as an indicator of systemic toxicity. For melanoma targeting studies, radiolabeled 2-thiouracil is injected into melanoma-bearing mice, and incorporation into tumor pigment is analyzed by evaluating melanin absorption at 350 nm and measuring PTCA yields after chemical degradation .

Absorption, Distribution and Excretion
Drug delivery to the fetus: Time to fetal presence: 10 minutes; Time to reach fetal/maternal concentration equilibrium: 40 minutes; Fetal/maternal concentration ratio of 1.0 /Excerpt from Table/
/In rats/ (35)S-labeled accumulation was faster and more extensive starting from smaller doses, indicating a saturable transport mechanism. Similar studies using (14)C-thiouracil showed radioactive accumulation in the thyroid tissue of both mother rabbits and fetuses…
…Whether using large doses (39 μmol) or small doses (1.2 μmol) of (35)S-thiouracil, the total radioactivity and accumulation of unmetabolized (35)S-thiouracil in thyroid tissue were significantly higher than in rat plasma compared to plasma levels.
Thiouracil can be absorbed through the rat gastrointestinal tract… After intravenous injection of 5 mg thiouracil in rats, only 30% of the thiouracil was recovered from the cadaver 3 hours later, with the remainder being trace amounts. 24 hours later.
For more data on the absorption, distribution and excretion (complete) of 2-thiouracil (7 metabolites), please visit the HSDB record page.

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Metabolism/Metabolites
In homogenized liver preparations from female Holtzman rats, 28-35% of thiouracil was metabolized within 3 hours. The degradation pathway of thiouracil is presumed to be: uracil; β-urea propionic acid, which is further metabolized to β-alanine; ammonia and carbon dioxide…

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The stability of thiouracil under the assay conditions was evaluated by high performance liquid chromatography (HPLC). Incubation of TU (500 μM) with high concentrations of BH4 (up to 100 μM) did not result in significant degradation, confirming its stability during enzyme activity assays. [1]

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Pharmacokinetic data for 2-thiouracil indicates that antithyroid drugs of this class tend to have short half-lives and are primarily excreted in urine . The absorption, distribution, metabolism, and excretion (ADME) properties vary depending on individual factors and the specific derivative. The more lipophilic analogue, 6-propyl-2-thiouracil (propylthiouracil), demonstrates greater cellular penetration compared to the parent compound . In melanotic melanocytes exposed to propylthiouracil at high concentrations (40 μg/10⁶ cells), appreciable accumulation was observed, which paralleled its greater toxicity compared to 2-thiouracil . The compound can be detected in biological fluids using sensitive analytical methods such as LC-MS/MS, with recent methods achieving detection limits of 10 μg/L in bovine urine .

2-Thiouracil is classified as a hazardous substance with potential carcinogenicity. According to the International Agency for Research on Cancer (IARC), 2-thiouracil can cause cancer . Under the OSHA Hazard Communication Standard (29 CFR 1910.1200), it falls under Carcinogenicity Category 2, indicating it is suspected of causing cancer . Known adverse effects include blood dyscrasias (hematological abnormalities), and the compound is suspected of teratogenicity and carcinogenicity . In animal studies, administration of 2-thiouracil can induce arteriosclerosis . However, when coordinated as Ru(II) complexes, the systemic toxicity appears tolerable, as treated mice showed no significant body weight changes or major organ damage . Safety precautions include obtaining special instructions before use, wearing appropriate personal protective equipment, storing in a locked-up area, and disposing through approved waste disposal protocols . The compound is strictly for research use only, not for human therapeutic applications without proper authorization.

[1]. 2-thiouracil is a selective inhibitor of neuronal nitric oxide synthase antagonising tetrahydrobiopterin-dependent enzyme activation and dimerisation. FEBS Lett, 2000. 485(2-3): p. 109-12.

[2]. Mechanism of selective incorporation of the melanoma seeker 2-thiouracil into growing melanin. J Med Chem, 1996. 39(26): p. 5192-201.

According to the International Agency for Research on Cancer (IARC) of the World Health Organization, thiouracil is potentially carcinogenic. Thiouracil is a nucleobase analog, a derivative of uracil in which the oxygen group at the C-2 position is replaced by a thio group. It is both an antithyroid drug and a metabolite. It is a thiocarbonyl compound and also a nucleobase analog. It is functionally related to uracil. 2-Thiouracil has been reported in Euglena gracilis, and relevant data are available. Thiouracil is a thiouracil-containing compound. As a known antithyroid drug and a highly selective nitric oxide synthase (NOS) inhibitor, thiouracil can also covalently bind to dopaquinone, produced by tyrosinase-catalyzed tyrosine oxidation, thereby selectively accumulating in newly synthesized melanin in overactive melanocytes and providing a method for locating melanoma cells. (NCI04) It is found in the seeds of Brassica and Cruciferae plants. Thiouracil was once used as an antithyroid drug, a coronary vasodilator, and a treatment for congestive heart failure, but its use has been largely superseded by other drugs. It is known to cause blood disorders and is suspected of being teratogenic and carcinogenic.

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Mechanism of Action
Antithyroid drugs primarily inhibit the formation of thyroid hormones by interfering with the organification of iodine. This means they interfere with the oxidation of iodide ions, but the detailed mechanism is difficult to elucidate due to incomplete understanding of the thyroid iodine oxidation system. /Antithyroid Drugs/
Thiodamide derivatives do not have permanent effects on the thyroid gland but inhibit hormone synthesis and secretion until spontaneous remission occurs during the disease process. /Thiodamide Derivatives/

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Therapeutic Uses
Antimetabolites; Antithyroid Drugs; Vasodilators
Thiodamide has been reported as an antithyroid drug in human medicine and for the treatment of angina and congestive heart failure… However, there is no evidence that thiodamide is currently used for these purposes in the United States.
Treatment for hyperthyroidism; angina pectoris; congestive heart failure / Previous use in China: USA /
Drugs (Veterinary): Thyroid inhibitors; used to treat hyperthyroidism and promote weight gain
Drug Warnings
The main disadvantage of antithyroid drug treatment is the high relapse rate after discontinuation. /Antithyroid Drugs/
Patients should immediately report sore throat or fever, which is often a precursor to agranulocytosis. If mild granulocytopenia is found, it may be a sign of thyrotoxicosis or the first symptom of this dangerous drug reaction. Caution and frequent white blood cell counts are necessary in this case. Thiamine derivatives: Women taking these drugs should not breastfeed their infants. Antithyroid drugs: It is unclear at what stage of pregnancy treatment will affect the fetus (fetal goiter and hypothyroidism), but in some cases, medication is not started until two-thirds of the pregnancy. Even later maternal medication may cause this. /Antithyroid Drugs/
For more drug warnings (full) data on 2-thiouracil (6 of them), please visit the HSDB record page.

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2-thiouracil is an approved antithyroid drug and is also an investigational drug for the detection and targeting of metastatic melanoma. Its antithyroid and melanoma-targeting properties are related to its inhibitory effects on enzymes such as thyroid iodine peroxidase, myeloperoxidase, eosinophil peroxidase, and tyrosinase. [1]
Given the presence of NOS activity in the thyroid gland, the newly discovered TU’s properties as a selective nNOS inhibitor may help in understanding its antithyroid effects. It also represents a potential lead compound for the development of neuroprotective agents. [1]
This inhibition competes with L-arginine. Increasing the concentration of the cofactor tetrahydrobiopterin (BH4) can reverse this inhibition, suggesting that the compound interacts with or is adjacent to the BH4 binding site. The sulfur atom in the thiourea moiety is crucial for reactivity, since both uracil (which lacks sulfur) and thiourea are ineffective. [1]

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- Mechanism of Selective Incorporation: 2-Thiouracil accumulates in melanoma by chemically reacting with reactive quinonoid intermediates generated during active melanogenesis. Unlike sulfhydryl compounds like cysteine, which react mainly at the 5-position of dopaquinone, TU adds almost exclusively at the 6-position to form 6-S-TU-DOPA. TU also reacts with downstream indolequinones from DHI (at C-2 and C-3) and DHICA (at C-4). The resulting TU-indole adducts can then be incorporated into the growing melanin polymer via oxidative copolymerization. [2]
- Effect on Melanin Structure: Incorporation of TU significantly alters the properties of the resulting melanin, causing a decrease in A₃₅₀ absorbance, altered yields of degradation products (PTCA and PDCA), and changes in solubility and color compared to normal eumelanin. [2]
- Clinical Potential: TU and related thiourylene compounds have been investigated as melanoma-seeking agents for diagnostic imaging (e.g., with radioiodine) and for potential boron neutron capture therapy (BNCT) due to their selective accumulation in melanoma tissues. [2]
- Key Isolated Compounds: The study isolated and characterized several novel TU-indole adducts, including 1 (C-3 substituted DHI-TU), 2 (C-2 substituted DHI-TU), 3 (a 4,4'-biindolyl dimer of 2), 4 (a trimer of DHI with two TU units), 5 (C-4 substituted DHICA-TU), 6 (a 3,4'-biindolyl dimer of 5), and 7 (a trimer of DHICA with one TU unit). Full NMR and MS characterization was provided for these compounds. [2]

C4H4N2OS

128.15236

128.004

141-90-2

2-Thiouracil-13C,15N2

1269845

White to off-white solid powder

1.5±0.1 g/cm3

337.2ºC at 760mmHg

>300 °C(lit.)

157.7ºC

5.45E-05mmHg at 25°C

1.678

-0.28

2

2

0

8

163

0

C1=CNC(=S)NC1=O

ZEMGGZBWXRYJHK-UHFFFAOYSA-N

InChI=1S/C4H4N2OS/c7-3-1-2-5-4(8)6-3/h1-2H,(H2,5,6,7,8)

2-sulfanylidene-1H-pyrimidin-4-one

2-thiouracil; Thiouracil; 141-90-2; Deracil; Antagothyroid;

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 : ~50 mg/mL (~390.17 mM)
H2O : ~0.67 mg/mL (~5.23 mM)

Solubility in Formulation 1: ≥ 3.75 mg/mL (29.26 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 37.5 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: ≥ 3.75 mg/mL (29.26 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 37.5 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.)