Preparation method of stable isotope labeled methotrexate internal standard reagent

A stable isotope, methotrexate technology, applied in the directions of isotope introduction of heterocyclic compounds, isotope introduction of organic compounds, organic chemical methods, etc., can solve the problems of high price and rare raw materials, and achieve low cost, short synthesis steps, and reaction mild conditions

Active Publication Date: 2022-05-10
谱同生物医药科技常州有限公司
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

The synthetic method used in this literature 13 C-labeled 6-bromoethylpteridine-2,4-diamine is a raw material, which is rare and very expensive, and is not suitable for commercial application

Method used

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  • Preparation method of stable isotope labeled methotrexate internal standard reagent
  • Preparation method of stable isotope labeled methotrexate internal standard reagent
  • Preparation method of stable isotope labeled methotrexate internal standard reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1: Diethyl N-(4-methylaminobenzoyl)-L-glutamate (N-methyl-D 3 )preparation

[0059]

[0060] In a 100mL three-necked flask, add deuterated methylbenzenesulfonate (1.42g, 7.5mmol), N-(4-aminobenzoyl)-L-diethyl glutamate (2.74g, 8.25mmol) , Tetrabutylammonium bromide (12.46g, 37.5mmol), 10mL of 20% aqueous potassium carbonate solution, N, N-dimethylformamide (30mL); reacted at 70°C for 20 hours; TLC traced the reaction of the reaction materials to complete Afterwards, the reaction solution was cooled to room temperature, 200 mL of water was added, extracted with ethyl acetate (3×100 mL), the organic phases were combined, washed successively with saturated sodium bicarbonate solution and saturated sodium chloride solution, and dried by adding anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and the residue was subjected to column chromatography to obtain stable isotope labeled N-(4-methylaminobenzoyl)-L-glutamic acid diethyl ester...

Embodiment 2

[0061] Example 2: Diethyl N-[4-[[(2,4-diamino-6-pteridine)methyl]methylamino]benzoyl]-L-glutamate (N-methyl-D 3 ) preparation

[0062]

[0063] In a 100mL three-neck flask, add stable isotope labeled N-(4-methylaminobenzoyl)-L-glutamic acid diethyl ester (N-methyl-D 3 ) (0.63g, 1.84mmol), 6-(bromomethyl)-2,4-pteridine diamine hydrobromide (0.69g, 1.84mmol), N, N-dimethylacetamide (20mL), Diisopropylethylamine (0.71g, 5.52mmol); reacted at 50°C for 16 hours; TLC tracked the reaction raw materials after the reaction was complete, distilled off the solvent under reduced pressure, added 20mL of water, and washed with ethyl acetate (3 × 30mL ) extraction, combined organic phases, washed with saturated sodium bicarbonate solution successively, washed with saturated sodium chloride solution, added anhydrous sodium sulfate to dry, evaporated under reduced pressure to remove the solvent, and the residue obtained N-[4-[[ (2,4-Diamino-6-pteridine)methyl]methylamino]benzoyl]-L-glutam...

Embodiment 3

[0064] Embodiment 3: methotrexate (N-methyl-D 3 ) preparation

[0065]

[0066] In a 50mL three-necked flask, add N-[4-[[(2,4-diamino-6-pteridine)methyl]methylamino]benzoyl]-L-glutamic acid diethyl ester (N-form Base-D 3 ) (0.17g, 0.33mmol), methanol (5mL), sodium hydroxide (0.04g, 1mmol); and reacted at 30°C for 5 hours; TLC traced that the reaction raw materials were completely reacted, the solvent was distilled off under reduced pressure, and 10mL of water was added, Regulate the pH value to 7 with hydrochloric acid of 1mol / L, stand and refrigerate and then crystallize; filter and collect the precipitated yellow crystals, obtain stable isotope labeled methotrexate (N-methyl-D 3 ) (0.12g, yield 80%). 1 H NMR (DMSO-D 6 , 600MHz): δ12.33(brs, 2H), 8.58(s, 1H), 8.19(d, J=6Hz, 1H), 7.74(d, J=6Hz, 1H), 7.67(brs, 1H), 7.45 (brs, 1H), 6.83(d, J=6Hz, 2H), 6.63(brs, 2H), 4.78(s, 2H), 4.37~4.33(m, 1H), 2.32(t, J=6Hz, 2H) , 2.08~2.02(m, 1H), 1.95~1.88(m, 1H), see attached Fi...

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Abstract

The invention relates to a preparation method of a stable isotope labeled methotrexate internal standard reagent for monitoring the blood concentration of a clinical treatment drug, and belongs to the field of research and development of standard substances for monitoring the blood concentration of the clinical treatment drug. The preparation method comprises the following steps: by taking stable isotope labeled methyl p-toluenesulfonate as a raw material, carrying out addition, condensation and hydrolysis, and finally separating and purifying to obtain the stable isotope labeled methotrexate. The process route has the advantages that raw materials required for synthesis are simple and easy to obtain, reaction conditions are mild, a highly toxic methylation reagent is not used, and a final product is high in total yield and easy to separate and purify. The chemical purity of the final product stable isotope labeled methotrexate can reach 99% or above, and the isotope abundance can reach 99% or above. The stable isotope labeled methotrexate internal standard reagent prepared by the preparation method can meet the technical requirements of the stable isotope labeled internal standard reagent required by a liquid chromatography-tandem mass spectrometry method for monitoring the blood concentration of clinical treatment drugs.

Description

technical field [0001] The invention relates to a preparation method of a stable isotope-labeled methotrexate internal standard reagent for monitoring blood drug concentration of clinical therapeutic drugs, and belongs to the field of research and development of standard substances for monitoring blood drug concentration of clinical therapeutic drugs. Background technique [0002] Methotrexate (Methotrexate, MTX) is an anti-folate antineoplastic drug, which mainly hinders the synthesis of tumor cells by inhibiting dihydrofolate reductase, and inhibits the growth and reproduction of tumor cells. It is widely used in the treatment of Choriocarcinoma, malignant hydatidiform mole, various types of acute leukemia, breast cancer, small cell lung cancer, head and neck cancer, gastrointestinal cancer, cervical cancer and malignant lymphoma, etc. Methotrexate is one of the commonly used drugs for the clinical treatment of children with acute lymphoblastic leukemia at the present stag...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D475/08C07B59/00G01N30/06
CPCC07D475/08C07B59/002G01N30/06C07B2200/05C07B2200/07G01N2030/045Y02P20/55
Inventor 王玮李珂珂李宇孟潘黎东
Owner 谱同生物医药科技常州有限公司
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