MTX/methotrexate analog; DHFR/dihydrofolate reductase; anticancer
The primary targets of NSC341076 include dihydrofolate reductase (DHFR) and the efflux pump ABCG2. It acts as a DHFR inhibitor and is a substrate for ABCG2. Methotrexate Triglutamate is a metabolite of methotrexate and retains the ability to inhibit DHFR. ABCG2 is a plasma membrane efflux pump that is able to confer resistance to several anticancer agents, including mitoxantrone, camptothecins, anthracyclines, and flavopiridol.

ABCG2 is a plasma membrane efflux pump that is able to confer resistance to several anticancer agents, including mitoxantrone, camptothecins, anthracyclines, and flavopiridol. The antimetabolite methotrexate (MTX) was inferred recently to be an additional substrate of the pump based on the analysis of ABCG2-overexpressing cell lines. However, the transport characteristics of the pump with regard to this agent have not been determined. In addition, physiological substrates of ABCG2 have not been identified. Here we examine the in vitro transport properties of the pump using membrane vesicles prepared from HEK293 cells transfected with ABCG2 expression vector. In so doing it is shown that MTX is a high capacity low affinity substrate of the pump, with K(m) and V(max) values of 1.34 +/- 0.18 mM and 687 +/- 87 pmol/mg/min, respectively. Unlike previously characterized multidrug resistance protein family members, ABCG2 is also able to transport MTX diglutamate and MTX triglutamate. However, addition of even one more glutamyl residue is sufficient to completely abrogate ABCG2-mediated transport. By contrast with the wild-type protein (ABCG2-R482), two ABCG2 variants that have been identified in drug selected cell lines, R482T and R482G, were unable to transport MTX to any extent. Similarly, folic acid was subject to efflux by the wild-type protein but not by the two mutants. However, transport of the reduced folate leucovorin was not detected for either the wild-type or the mutant proteins. Finally, it is shown that ABCG2 is capable of transporting E(2)17betaG with K(m) and V(max) values of 44.2 +/- 4.3 micro M and 103 +/- 17 pmol/mg/min, respectively. These results indicate that ABCG2 is a component of the energy-dependent efflux system for certain folates and antifolates, but that its transport characteristics with respect to polyglutamates and reduced folates are not identical to those of multidrug resistance protein family members. In addition, it is demonstrated that R482 mutations observed in drug-resistant cell lines have profound effects on the in vitro transport properties of the pump[1].
In vitro, NSC341076 is a DHFR inhibitor. It has potential anti-inflammatory activity. The compound is a substrate for ABCG2, and its efflux by this pump can contribute to drug resistance. Its activity is typically measured using DHFR enzyme assays or cell-based assays that assess its anti-proliferative or anti-inflammatory effects.

In vivo, NSC341076 is a metabolite of methotrexate. As a DHFR inhibitor, it contributes to the pharmacological effects of methotrexate, including its antineoplastic, antirheumatic, and anti-inflammatory activities.

In vitro enzyme/receptor binding assays for NSC341076 involve DHFR inhibition assays using purified DHFR enzyme. The compound's inhibitory activity is measured by monitoring the reduction of dihydrofolate to tetrahydrofolate in the presence of NADPH. IC50 values are determined from dose-response curves. ABCG2 transport assays can be performed to assess the compound's interaction with this efflux pump.

In vitro cellular assays for NSC341076 are conducted in cancer cell lines or cells expressing ABCG2. Cells are treated with varying concentrations of NSC341076, and cell proliferation is measured using MTT, CellTiter-Glo, or colony formation assays. The compound's interaction with ABCG2 is assessed by measuring the intracellular accumulation of the compound in the presence and absence of ABCG2 inhibitors.

In vivo animal studies for NSC341076 are not typically performed as a standalone compound, as it is a metabolite of methotrexate. However, its effects would be similar to those of methotrexate, which is studied in animal models of cancer, arthritis, and lupus erythematosus.

Pharmacokinetic properties of NSC341076 indicate that it has a molecular weight of 712.67 and a molecular formula of C30H36N10O11. As a metabolite of methotrexate, its pharmacokinetics are related to those of the parent drug. The compound is typically stored as a powder at appropriate conditions to maintain stability.

The toxicological profile of NSC341076 is similar to that of methotrexate, which can cause myelosuppression, hepatotoxicity, and gastrointestinal toxicity. As a DHFR inhibitor, it can affect rapidly dividing cells, leading to these side effects.

[1]. Transport of methotrexate, methotrexate polyglutamates, and 17beta-estradiol 17-(beta-D-glucuronide) by ABCG2: effects of acquired mutations at R482 on methotrexate transport. Cancer Res. 2003 Jul 15;63(14):4048-54.

NSC341076 is a metabolite of methotrexate and a DHFR inhibitor. It is also known as Methotrexate Triglutamate. The compound has potential anti-inflammatory activity. NSC341076 is not approved for clinical use and is available from research chemical suppliers for research applications.

C30H36N10O11

712.67

712.257

C, 50.56; H, 5.09; N, 19.65; O, 24.69

41600-14-0

456144

Typically exists as solid at room temperature

1.54g/cm3

1.683

1.302

9

18

19

51

1270

3

CN(CC1=CN=C2C(=N1)C(=NC(=N2)N)N)C3=CC=C(C=C3)C(=O)NC(CCC(=O)NC(CCC(=O)NC(CCC(=O)O)C(=O)O)C(=O)O)C(=O)O

SBFRVUOTZGQNGW-FHWLQOOXSA-N

InChI=1S/C30H36N10O11/c1-40(13-15-12-33-25-23(34-15)24(31)38-30(32)39-25)16-4-2-14(3-5-16)26(45)37-19(29(50)51)7-10-21(42)35-17(27(46)47)6-9-20(41)36-18(28(48)49)8-11-22(43)44/h2-5,12,17-19H,6-11,13H2,1H3,(H,35,42)(H,36,41)(H,37,45)(H,43,44)(H,46,47)(H,48,49)(H,50,51)(H4,31,32,33,38,39)/t17-,18-,19-/m0/s1

(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(2,4-diaminopteridin-6-yl)methyl-methylamino]benzoyl]amino]butanoyl]amino]butanoyl]amino]pentanedioic acid

NSC-341076; NSC 341076; NSC341076; 41600-14-0; NSC 341076; L-Glutamic acid, N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-gamma-glutamyl-L-gamma-glutamyl-; L-Glutamic acid, N-(N-(4-(((2,4-diamino-6-pteridinyl)methyl)methyl-; (2S)-2-[[(4S)-4-Carboxy-4-[[(4S)-4-carboxy-4-[[4-[(2,4-diaminopteridin-6-yl)methyl-methylamino]benzoyl]amino]butanoyl]amino]butanoyl]amino]pentanedioic acid; L-Glutamic acid, N-[N-[4-[[(2,4-diamino-6-pteridinyl)methyl]methyl-; CHEMBL2074970; NSC341076

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)

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

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.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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).

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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)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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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).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)