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| Other Sizes |
| Targets |
5-Chlorouracil targets nucleic acid metabolism through multiple mechanisms. As a halogenated uracil analog, its primary mechanism of action involves incorporation into RNA and, following metabolic conversion to its deoxyribonucleotide form, into DNA, disrupting normal nucleic acid synthesis and function . This incorporation leads to inhibition of thymidylate synthase and induces replication errors, making it valuable for studying cell cycle arrest and apoptosis. Additionally, certain 6-substituted derivatives of 5-chlorouracil have been identified as selective and potent competitive inhibitors of thymidine phosphorylase (TP), an enzyme with angiogenic activity, with the analog 6-(2-aminoethyl)amino-5-chlorouracil (AEAC) exhibiting a Ki of 165 nM . These inhibitors specifically target TP without affecting purine nucleoside phosphorylase (PNP) or uridine phosphorylase (UP) at concentrations up to 1 mM .
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| ln Vitro |
5-Chlorouracil is a derivative of pyrimidine nucleoside base uracil that has been chlorinated. It becomes the mutagenic and genotoxic chlorodeoxyuridine in vivo. One way to chlorinate uracil is at position 5, where it combines with myeloperoxidase, peroxide, and chloride to form hypochlorous acid. Two ways to study uracil in vitro and in vitro with phorbol 12-Myristate 13-acetate-stimulated human neutrophils were discovered in inflammatory human exudates that were isolated from superficial infection sites. Exudate extracted from the site of inflammation showed elevated 5-chlorouracil levels in both patient-derived human atherosclerotic aorta tissue and a rat model of carrageenan-induced inflammation.
In vitro studies have demonstrated that 5-chlorouracil exhibits antiproliferative effects on ocular fibroblasts and conjunctival cells, with studies comparing its activity to 5-fluorouracil (5-FU) . While 5-FU showed higher antiproliferative and toxic effects than 5-CU, 5-chlorouracil induced predominantly apoptosis in treated cells, whereas 5-FU induced necrosis . The apoptosis induced by 5-CU is driven through a non-caspase-dependent pathway involving apoptosis-inducing factor (AIF) and LEI/L-DNase II . In enzyme inhibition studies, thymidine phosphorylase (TP) induced human umbilical vein endothelial cell (HUVEC) migration in modified Boyden chamber assays, and this action could be abrogated by 5-chlorouracil-derived TP inhibitors . The inhibitory effects were specific for TP, as these compounds had no effect on VEGF-induced chemotaxis . |
| ln Vivo |
In vivo studies have demonstrated that controlled delivery of 5-chlorouracil using poly(ortho esters) (POE) has a longstanding effect on intraocular pressure (IOP) reduction after glaucoma-filtering surgery in rabbit eyes . At 34 days after surgery, the mean IOP in the POE/5-CU-treated group was 83% of baseline, compared to only 40% in the POE/5-FU-treated group, indicating more sustained IOP reduction with 5-CU . Histologic analysis showed evidence of functioning blebs in the POE-5-CU-treated eyes along with preserved structure of the conjunctiva epithelium, suggesting that slow-release 5-CU may be beneficial for preventing bleb closure in patients undergoing complicated trabeculectomy . In anti-tumor studies, chromium(III) complexes of 5-chlorouracil demonstrated significant activity against P815 murine mastocytoma, while aluminum(III) complexes showed poor activity, suggesting that metal complexation can modulate the biological activity of the compound .
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| Enzyme Assay |
A highly sensitive and specific gas chromatography-mass spectrometry (GC-MS) assay has been developed for the detection of 5-chlorouracil as a marker of DNA damage . In this assay, DNA is enzymatically digested using nuclease P1 (3 μl of 3.3 mg/ml, 65°C for 10 min), followed by alkaline phosphatase (2 μl of 4 units/μl, 37°C for 1 hour) to generate nucleosides . Thymidine phosphorylase (1 unit) is then added to release free 5-chlorouracil and thymine from the nucleosides in the presence of 0.2 M potassium phosphate buffer (pH 7.2) at 37°C for 1 hour . Following extraction with ethyl acetate, the freed 5-chlorouracil is derivatized with 3,5-bis(trifluoromethyl)benzyl bromide (BTFMBzBr) at 37°C for 25 minutes, and the derivative is detected by negative chemical ionization mass spectrometry with selective ion monitoring at m/z 371 . This assay achieves a limit of detection of approximately 0.2 fmol on column and can simultaneously detect other halogenated uracils .
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| Cell Assay |
Cellular assays for 5-chlorouracil have been established using human HeLa cells and murine embryo C3H10T1/2 cells . Cells are seeded with approximately 60-70% confluence 24 hours before treatment. For HOCl-induced damage studies, cells are incubated with 10-40 μM of HOCl in Hank's Balanced Salt Solution (HBSS) at 37°C for 10-30 minutes and then immediately harvested, or after HOCl treatment, cells are replenished with DMEM containing 5% FBS and further incubated for 24 hours before harvesting . For antiproliferative and toxicity evaluation, rabbit Tenon fibroblasts and human conjunctival cells are incubated with various 5-chlorouracil concentrations (typically ranging from 0.1 to 1000 μM). Cell viability is assessed at 24 and 72 hours using monotetrazolium (MTT), neutral red, and Hoechst tests, and cell counting . Cell death mechanisms are evaluated using TUNEL assay, annexin V binding, and immunohistochemistry for apoptosis-inducing factor (AIF) and LEI/L-DNase II .
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| Animal Protocol |
An established in vivo protocol for 5-chlorouracil involves the rabbit trabeculectomy model for glaucoma research . Pigmented rabbits undergo trabeculectomy surgery, and 200 microliters of poly(ortho esters) (POE) loaded with 1% wt/wt 5-chlorouracil is injected into the subconjunctival space immediately after surgery . Intraocular pressure (IOP) and bleb persistence are monitored for up to 150 days post-operatively using tonometry. In the carrageenan-induced inflammation model in rats, air pouches are created by subcutaneous injection of sterile air into the intrascapular area of male Wistar rats (250-330 g), and inflammation is induced by carrageenan injection. Exudate fluid is isolated from the inflammation site, and chlorinated nucleosides, including 5-chlorouracil, are detected using GC-MS as described above .
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| ADME/Pharmacokinetics |
Specific pharmacokinetic data for 5-chlorouracil is limited in the available literature . However, based on its structural similarity to 5-fluorouracil, which is known to have erratic oral bioavailability and a short biological half-life, 5-chlorouracil likely exhibits comparable absorption, distribution, metabolism, and excretion characteristics . The compound shows limited water solubility and is typically formulated for controlled delivery applications using polymer delivery systems such as poly(ortho esters) (POE) . For research applications, 5-chlorouracil is stable as a powder at room temperature and can be stored under standard laboratory conditions.
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| Toxicity/Toxicokinetics |
According to available safety data, 5-chlorouracil is classified as an irritant that may cause eye and skin irritation upon direct contact . In mutagenicity studies, the compound has shown positive results in Escherichia coli mutation test systems at a concentration of 50 mg/L for 1 hour . In comparative in vitro toxicity studies, 5-chlorouracil exhibited a less toxic profile than 5-fluorouracil, inducing predominantly apoptosis rather than necrosis . In animal studies using the rabbit trabeculectomy model, POE/5-CU treatment showed preserved conjunctival epithelium structure, indicating acceptable local tolerability . Ecological data indicates that 5-chlorouracil is slightly hazardous to water and should not be released into groundwater, or sewage systems without proper treatment . The compound is strictly intended for research use only and not for human therapeutic or diagnostic applications without appropriate authorization.
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| References |
[1]. https://pubchem.ncbi.nlm.nih.gov/compound/15758
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| Additional Infomation |
5-Chlorouracil is an organochlorine compound formed by replacing the chlorine at the 5-position of the uracil molecule. It is functionally related to uracil.
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| Molecular Formula |
C4H3CLN2O2
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|---|---|
| Molecular Weight |
146.53
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| Exact Mass |
145.988
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| CAS # |
1820-81-1
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| PubChem CID |
15758
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| Appearance |
White to off-white solid powder
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| Density |
1.6±0.1 g/cm3
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| Melting Point |
>300 °C(lit.)
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| Flash Point |
203.9ºC
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| Vapour Pressure |
1.98E-07mmHg at 25°C
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| Index of Refraction |
1.587
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| LogP |
-0.52
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
2
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| Rotatable Bond Count |
0
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| Heavy Atom Count |
9
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| Complexity |
199
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| Defined Atom Stereocenter Count |
0
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| SMILES |
ClC1=C([H])N([H])C(N([H])C1=O)=O
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| InChi Key |
ZFTBZKVVGZNMJR-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C4H3ClN2O2/c5-2-1-6-4(9)7-3(2)8/h1H,(H2,6,7,8,9)
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| Chemical Name |
5-chloro-1H-pyrimidine-2,4-dione
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| Synonyms |
5-CHLOROURACIL; 1820-81-1; 2,4(1H,3H)-Pyrimidinedione, 5-chloro-; 5-chloropyrimidine-2,4(1H,3H)-dione; 7LQ4V03RNY;
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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 Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| 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 | 6.8245 mL | 34.1227 mL | 68.2454 mL | |
| 5 mM | 1.3649 mL | 6.8245 mL | 13.6491 mL | |
| 10 mM | 0.6825 mL | 3.4123 mL | 6.8245 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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