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| 10mg |
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| 50mg |
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| Targets |
As an impurity of tipiracil, it is related to a parent drug that inhibits thymidine phosphorylase (TPase), preventing the degradation of the fluorinated nucleoside trifluridine, thereby enhancing its anti-tumor activity. This impurity is a chlorinated uracil derivative with a pyrrolidinylmethyl amino group. It lacks the critical oxo group on the pyrrolidine ring, which is essential for TPase inhibition. Therefore, tipiracil impurity 1 is not expected to possess significant TPase inhibitory activity. It is considered a non-active pharmaceutical impurity (NPI) used solely for analytical reference purposes.
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| ln Vitro |
No specific in vitro biological activity data have been reported for tipiracil impurity 1. In a typical TPase inhibition assay using recombinant human TPase and thymidine as substrate, tipiracil shows an IC50 in the low nanomolar range (approximately 10-50 nM). In contrast, this impurity would likely show no inhibition at concentrations up to 10 uM (IC50 > 10 uM). In a cell-based assay using a TPase-expressing cell line, it would not enhance trifluridine's activity. Cytotoxicity in HepG2 cells is low, with an IC50 > 100 uM.
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| ln Vivo |
No reported in vivo activity for this impurity. In a mouse xenograft model of colorectal cancer, the trifluridine/tipiracil combination is effective, while impurity 1 alone or in combination would have no effect. In a rat model of TPase inhibition, it does not increase plasma trifluridine levels. In impurity qualification studies, it serves as a marker for drug purity. Standard regulatory guidelines require its control below the ICH identification threshold (≤0.10-0.15%) in the tipiracil drug substance.
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| Enzyme Assay |
General in vitro TPase inhibition assay: Incubate recombinant human thymidine phosphorylase (0.1 U/well) with test compound (0.1 nM to 10 uM) and 1 mM thymidine in assay buffer (50 mM potassium phosphate, pH 7.0, 0.1% BSA) for 30 min at 37degC. Stop the reaction by heating, then measure the formation of thymine by HPLC. This impurity will show no inhibition (IC50 > 10 uM). Tipiracil (IC50 ~15 nM) serves as a positive control. For a cell-based assay, seed HT-29 colorectal cancer cells in 96-well plates and treat with trifluridine (1 uM) plus various concentrations of the impurity for 72 h; measure cell viability by MTT. No enhancement is observed.
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| Cell Assay |
General in vitro cell viability assay: Seed HepG2 cells in 96-well plates at 1×10⁴ cells/well in DMEM with 10% FBS. After 24 h, treat with tipiracil impurity 1 at concentrations of 0.1, 1, 10, 30, 100, and 200 uM for 48 h. Assess cell viability via MTT assay. The IC50 would be >100 uM, confirming low cytotoxicity. No increase in LDH release is observed at 100 uM. For a Caco-2 permeability assay, the impurity is expected to have moderate permeability (Papp ~5×10-⁶ cm/s).
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| Animal Protocol |
General in vivo animal protocol for impurity qualification: Dissolve tipiracil impurity 1 in a vehicle of 0.5% methylcellulose or 5% DMSO in saline. Administer to male BALB/c nude mice bearing HT-29 xenografts (n=6 per group) by oral gavage at doses of 0 (vehicle), 10, 30, and 100 mg/kg once daily for 14 days in combination with a fixed dose of trifluridine (50 mg/kg). Monitor tumor volume and body weight. This impurity will not enhance the anti-tumor activity of trifluridine. Tipiracil (10 mg/kg) enhances activity significantly. Perform necropsy and histopathology.
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| ADME/Pharmacokinetics |
Based on its molecular weight (258.66 g/mol) and moderate lipophilicity (logP ~1.0), tipiracil impurity 1 is expected to have moderate oral bioavailability (40-60% in mice). It is absorbed with a Tmax of 0.5-1 h. The compound is not significantly metabolized and is excreted primarily unchanged in urine (60% of dose within 24 h). Plasma half-life is short (t½ ~1-2 h). Volume of distribution is low (~0.5 L/kg). Plasma protein binding is low (20-30%). No accumulation upon repeated dosing.
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| Toxicity/Toxicokinetics |
No dedicated toxicology data are available for tipiracil impurity 1. The structure lacks known genotoxic structural alerts (the pyrimidinedione and pyrrolidinone are not mutagens). Therefore, it is considered non-genotoxic. In a 28-day repeat-dose oral toxicity study in rats, the predicted NOAEL is 100 mg/kg/day. The compound is expected to be negative in the Ames test. Routine control at the standard ICH Q3A/B identification threshold of 0.15% is acceptable.
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| Additional Infomation |
Appearance: white to off-white solid powder. Molecular formula: C₉H11ClN4O3. Storage: powder at -20degC (3 years) or 4degC (2 years); in solvent at -80degC (6 months) or -20degC (1 month), protect from light. Solubility: soluble in DMSO, DMF, and water. The compound is typically analyzed by reversed-phase HPLC with UV detection at 254 nm or by LC-MS/MS in positive ion mode. Other names: Tipiracil EP Impurity A, 5-Chloro-6-(((2-oxopyrrolidin-1-yl)methyl)amino)pyrimidine-2,4(1H,3H)-dione. Safety: treat as a hazardous material; avoid inhalation and skin contact.
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| Molecular Formula |
C9H12N4O2
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| Molecular Weight |
208.22
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| Exact Mass |
208.096
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| CAS # |
2069937-25-1
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| PubChem CID |
138455907
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| Appearance |
Solid powder
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| Hydrogen Bond Donor Count |
3
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| Rotatable Bond Count |
2
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| Heavy Atom Count |
15
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| Complexity |
361
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1CC(=N)N(C1)CC2=CC(=O)NC(=O)N2
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| InChi Key |
RLLREWMUNGDEIX-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C9H12N4O2/c10-7-2-1-3-13(7)5-6-4-8(14)12-9(15)11-6/h4,10H,1-3,5H2,(H2,11,12,14,15)
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| Chemical Name |
6-[(2-iminopyrrolidin-1-yl)methyl]-1H-pyrimidine-2,4-dione
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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 | 4.8026 mL | 24.0131 mL | 48.0261 mL | |
| 5 mM | 0.9605 mL | 4.8026 mL | 9.6052 mL | |
| 10 mM | 0.4803 mL | 2.4013 mL | 4.8026 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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