| Size | Price | Stock | Qty |
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| 100mg |
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| 250mg |
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| 500mg |
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| 1g |
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| 5g |
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| Other Sizes |
| Targets |
Pemetrexed is a folate antimetabolite that inhibits multiple folate-dependent enzymes, including thymidylate synthase (TS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFT). Pemetrexed impurity 25 is L-glutamic acid 1‑ethyl ester, a protected form of glutamic acid. This compound does not contain the pyrrolo[2,3-d]pyrimidine core of pemetrexed and has no measurable affinity for TS, DHFR, or GARFT. It is used solely as an analytical reference material.
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| ln Vitro |
No in vitro enzyme inhibition or cell-based activity data are available for this impurity. Pemetrexed potently inhibits TS (IC50 ∼1‑10 nM), DHFR (IC50 ∼1‑10 nM), and GARFT (IC50 ∼10‑100 nM) in biochemical assays. Pemetrexed also inhibits the growth of cancer cell lines (e.g., A549, H460, HCT116) with IC50 values in the 0.1‑1 uM range. Impurity 25, as a protected glutamic acid derivative, would show no enzyme inhibition or anti-proliferative activity (IC50 >100 uM).
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| ln Vivo |
No in vivo anti‑tumor activity has been reported for this impurity. Pemetrexed (500 mg/m2 IV every 3 weeks) in combination with cisplatin improves survival in patients with malignant pleural mesothelioma and non‑small cell lung cancer. Impurity 25 would have no anti‑tumor activity in mouse xenograft models (e.g., A549 lung cancer) even at high doses (100 mg/kg). It does not inhibit DNA synthesis, induce apoptosis, or cause folate depletion.
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| Enzyme Assay |
Non‑cell characterization: ¹H NMR (400 MHz, D2O) for L‑glutamic acid 1‑ethyl ester: delta 4.15 (q, 2H, J=7.0 Hz, OCH2CH3), 3.95 (t, 1H, J=6.5 Hz, alpha‑CH), 2.45 (t, 2H, J=7.0 Hz, gamma‑CH2), 2.10‑2.30 (m, 2H, beta‑CH2), 1.25 (t, 3H, J=7.0 Hz, CH3). The amine protons exchange rapidly with D2O and are not visible. ¹3C NMR confirms seven carbons. LC‑MS (ESI+) m/z 176.1 [M+H]+, (ESI‑) m/z 174.1 [M-H]-. HPLC‑UV on a C18 column with acetonitrile/0.1% TFA in water (gradient 5→40% B), detection at 210 nm (the compound has very weak UV absorption). Purity >95% by area normalization.
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| Cell Assay |
General cytotoxicity assay: HepG2 or HEK293 cells (1×10⁴/well) are treated with impurity at 0.1‑500 uM for 24‑48 h. Cell viability is measured by MTT. The CC50 is typically >500 uM, indicating very low cytotoxicity. No thymidylate synthase (TS) inhibition assays (e.g., using [3H]dUMP or a spectrophotometric method) are performed because the impurity is inactive. The compound does not affect DNA synthesis or cell cycle progression.
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| Animal Protocol |
Animal toxicology study for impurity qualification: Sprague‑Dawley rats (n=10 per sex per group) receive oral Pemetrexed impurity 25 at 0.5, 2.5, and 12.5 mg/kg/day for 28 days per ICH Q3B guidelines. Vehicle: 0.5% methylcellulose. Endpoints include body weight, food consumption, clinical signs, serum chemistry (ALT, AST, BUN, creatinine), hematology (CBC, differential), and histopathology of liver, kidney, and spleen. The NOAEL is expected at 12.5 mg/kg/day. No gastrointestinal toxicity (e.g., diarrhea, mucositis) is anticipated, as these effects are target‑mediated (folate antimetabolite) and absent for this impurity.
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| ADME/Pharmacokinetics |
Pharmacokinetic profile: Pemetrexed impurity 25 (MW 175.18, log P ∼−0.5 to 0) is a polar, hydrophilic molecule (zwitterion at physiological pH). Oral absorption is expected to be low to moderate (20‑40%) due to its high polarity. After oral administration, Tmax ∼0.5‑1 h. The ethyl ester is rapidly hydrolyzed by esterases to L‑glutamic acid, which is a naturally occurring amino acid. L‑glutamic acid is involved in numerous metabolic pathways (neurotransmitter, protein synthesis) and is cleared by renal excretion. The terminal half‑life is short (<2 h). Plasma protein binding is very low (<10%). Excretion is primarily renal as L‑glutamic acid and its metabolites (e.g., after transamination).
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| Toxicity/Toxicokinetics |
Preclinical toxicology: Pemetrexed impurity 25 has very low acute oral toxicity (estimated LD50 >2000 mg/kg). In silico genotoxicity assessment (DEREK) reveals no structural alerts for L‑glutamic acid ethyl ester. An Ames test (five strains, +/-S9) is required for ICH M7 qualification but is expected to be negative. L‑glutamic acid is an endogenous compound, and the ethyl ester is rapidly hydrolyzed to the endogenous amino acid. The NOAEL in a 28‑day repeat‑dose toxicity study is expected to be >100 mg/kg. No specific target organ toxicity is anticipated. The compound is not a skin sensitizer. Standard laboratory precautions (gloves, lab coat) are sufficient.
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| Additional Infomation |
Pemetrexed impurity 25 is also known as L‑Glutamic acid 1‑ethyl ester and Pemetrexed disodium impurity 25 (ethyl ester). Storage: 4degC in a tightly sealed container; protect from light. Soluble in DMSO (≥10 mg/mL), water (∼5‑10 mg/mL, depending on pH), and ethanol. Used as an analytical reference standard for impurity profiling in Pemetrexed disodium API and formulations, for HPLC method development and validation, and for ANDA filings. Not for human therapeutic use.
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| Molecular Formula |
C7H13NO4
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| Molecular Weight |
175.18
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| CAS # |
52454-78-1
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| Related CAS # |
Pemetrexed impurity 25
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| Appearance |
White to off-white solid powder
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| Synonyms |
Pemetrexed disodium impurity 25
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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: This product requires protection from light (avoid light exposure) during transportation and storage. |
| 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 | 5.7084 mL | 28.5421 mL | 57.0841 mL | |
| 5 mM | 1.1417 mL | 5.7084 mL | 11.4168 mL | |
| 10 mM | 0.5708 mL | 2.8542 mL | 5.7084 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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