CN114456170B - Preparation method of stable isotope labeled methotrexate internal standard reagent - Google Patents
Preparation method of stable isotope labeled methotrexate internal standard reagent Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D475/00—Heterocyclic compounds containing pteridine ring systems
- C07D475/06—Heterocyclic compounds containing pteridine ring systems with a nitrogen atom directly attached in position 4
- C07D475/08—Heterocyclic compounds containing pteridine ring systems with a nitrogen atom directly attached in position 4 with a nitrogen atom directly attached in position 2
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- C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
- C07B59/002—Heterocyclic compounds
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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Abstract
The invention relates to a preparation method of a stable isotope labeled methotrexate internal standard reagent for monitoring blood concentration of a clinical therapeutic drug, belonging to the field of research and development of standard substances for monitoring blood concentration of a clinical therapeutic drug. The invention takes stable isotope labeled methyl tosylate as a raw material, and finally obtains stable isotope labeled methotrexate through addition, condensation and hydrolysis. The process route of the invention has the advantages of simple and easily obtained raw materials, mild reaction conditions, no use of highly toxic methylation reagent, high total yield of the final product and easy separation and purification. The chemical purity of the final product of the stable isotope labeled methotrexate can reach more than 99 percent, and the isotope abundance can reach more than 99 percent. 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 for monitoring the blood concentration of a clinical therapeutic drug.
Description
Technical Field
The invention relates to a preparation method of a stable isotope labeled methotrexate internal standard reagent for monitoring blood concentration of a clinical therapeutic drug, belonging to the field of research and development of standard substances for monitoring blood concentration of a clinical therapeutic drug.
Background
Methotrexate (MTX) is an antifolate antineoplastic agent, which is used for inhibiting synthesis of tumor cells mainly by inhibiting dihydrofolate reductase, and inhibiting growth and reproduction of tumor cells, and is widely used for treating chorionic epithelial cancer, malignant grape embryo, various acute leukemia, breast cancer, small cell lung cancer, head and neck cancer, digestive tract cancer, cervical cancer, malignant lymphoma, etc. Methotrexate is one of the common drugs for clinically treating acute lymphoblastic leukemia of children at the present stage, the conventional dose of methotrexate is not easy to penetrate through blood brain, blood eye and blood testosterone barriers, and large dose of methotrexate can penetrate through the three barriers, so that the concentration of cerebrospinal fluid and blood drugs is effectively improved, and the treatment effect is exerted; meanwhile, large-dose methotrexate is also used as a standardized diagnosis and treatment scheme for osteosarcoma, and has been widely used in clinical treatment. The methotrexate has large individual differences in vivo distribution, biotransformation, absorption and excretion, and if delayed excretion occurs, obvious adverse reactions such as acute kidney injury, bone marrow depression, mucositis, hepatotoxicity, gastrointestinal tract reaction and the like can be caused. The curative effect and adverse reaction of the methotrexate are closely related to the dosage, and the individual difference is large, and different patients generate completely different clinical curative effects and adverse reactions on the same medicament and the same dosage, so that the clinical monitoring of the blood concentration of the methotrexate is important to ensure the medication safety of the patients.
Currently, there are many patents for monitoring the in-vivo blood concentration of the medicine, such as the Chinese patent application for detection method of methotrexate (publication number: CN 112415117A), "method for rapidly measuring the blood concentration of methotrexate" (publication number: CN 110320302A), "method for detecting the concentration of methotrexate substances in erythrocytes (publication number: CN 112666273A) all adopt liquid chromatography tandem mass spectrometry combined with stable isotope internal standard method for quantification, and all use stable isotope labeled methotrexate-D 3 As an internal standard for its stable isotope.
At present, the methotrexate marked by stable isotopes is only provided by foreign standard substance companies, and can only be solved by import, and the application of the compound in the domestic clinical treatment medicine blood concentration monitoring is severely made. To date, stable isotope labeled methotrexate-D 3 Only Charles S. Elmore, of Merck, U.S. was equal to Synthesis of C published in 2002 2 H 3 ]methotrexate and[C 2 H 3 ]7-hydroxymethotrexate[J]Journal of Labelled Compounds and Radiopharmaceuticals,2002, 45 (1): 29-36. The synthetic method of the document is to directly carry out reductive amination by using deuterium water, deuterated hydrochloric acid, deuterium water solution of deuterium hydroxide, deuterium water solution of deuterated formaldehyde, cyano sodium boron deuteride and aminopterin; the route has a plurality of side reactions, all the required raw materials are isotope labeling reagents, the reaction system has strict requirements and the technologyThe difficulty is high, the raw materials are expensive, the byproducts of the reaction system are more, the final product is required to be separated by a preparation liquid chromatograph, the total yield is low, the product cost is high, and the isotope abundance of the final product is less than 98%.
Multiple sites 13 The synthesis of C-stable isotope labeled methotrexate is described in Cheung, H.T.Andrew et al, synthesis ofmulti- 13 C-enriched methotrexate for NMR studies ofdrug-enzyme interactions [J]Hetercycles, 1987, 25 (1): 507-514. The synthesis method uses 13 The C-marked 6-bromoethyl pteridine-2, 4-diamine is used as a raw material, is difficult to obtain and has very high price, and is not suitable for being used as a product for commercialization.
Therefore, there is a need to develop a preparation method of stable isotope labeled methotrexate with easily available raw materials, low cost, simple operation, mild reaction conditions, short reaction route, high total yield of the final product, easy separation and purification and undiluted isotope abundance, so as to meet the increasing application requirements of the compound in the aspect of monitoring the blood concentration of the clinical therapeutic drugs in China.
Disclosure of Invention
The invention aims to provide a preparation route method of stable isotope labeled methotrexate for monitoring blood concentration of clinical therapeutic drugs. The synthetic route method has the advantages of easily available raw materials, low cost, simple operation, mild reaction conditions, short reaction route, high total yield of the final product and undiluted isotope abundance.
The aim of the invention can be achieved by the following technical scheme: the chemical structure of the stable isotope Labeled Methotrexate internal standard reagent is shown as a formula (Labeled-methotrexa):
wherein X is 12 C or 13 C, Y is H or D, characterized in that the overall route of the preparation steps is:
wherein X is 12 C or 13 C, Y is H or D, and the preparation steps are as follows:
(1) Adding stable isotope labeled methyl p-toluenesulfonate in the presence of an organic solvent, alkali and a phase transfer catalyst, then adding N- (4-aminobenzoyl) -L-diethyl glutamate, and reacting for 12-48 hours at 30-120 ℃; after TLC tracking reaction raw materials are reacted completely, quenching a reaction mixed solution by water, filtering, extracting a filtrate by using an organic solvent, combining organic phases, washing by using a saturated sodium bicarbonate solution and a saturated sodium chloride solution in sequence, adding a drying agent for drying, distilling under reduced pressure to remove the solvent, and carrying out column chromatography on residues to obtain stable isotope labeled N- (4-methylaminobenzoyl) -L-diethyl glutamate;
the reaction of this step is as follows:
wherein the chemical structure of the stable isotope labeled methyl tosylate is shown as a formula (I), and the chemical structure of the stable isotope labeled N- (4-methylaminobenzoyl) -L-diethyl glutamate is shown as a formula (II), wherein X is 12 C or 13 C, Y is H or D;
(2) Dissolving stable isotope labeled N- (4-methylaminobenzoyl) -L-glutamic acid diethyl ester in an organic solvent, adding 6- (bromomethyl) -2, 4-pteridine diamine hydrobromide and an acid binding agent, and reacting for 12-48 hours at 20-80 ℃; after TLC tracking reaction raw materials are reacted completely, quenching the reaction mixed solution by water, filtering, extracting the filtrate by an organic solvent, combining organic phases, washing by saturated sodium bicarbonate solution and saturated sodium chloride solution in sequence, adding a drying agent for drying, distilling under reduced pressure to remove the solvent, and obtaining stable isotope labeled N- [4- [ [ (2, 4-diamino-6-pteridine) methyl ] methylamino ] benzoyl ] -L-diethyl glutamate from residues by column chromatography;
the reaction of this step is as follows:
wherein the stable isotope labels N- [4- [ [ (2, 4-diamino-6-pteridine) methyl]Methylamino group]Benzoyl compounds]The chemical structure of the-L-diethyl glutamate is shown as a formula (III), wherein X is 12 C or 13 C, Y is H or D;
(3) Dissolving stable isotope labeled N- [4- [ [ (2, 4-diamino-6-pteridine) methyl ] methylamino ] benzoyl ] -L-glutamic acid diethyl ester in an organic solvent, adding inorganic base, and reacting for 2-12 hours at 20-100 ℃; after TLC tracking reaction raw materials are reacted completely, filtering a reaction solution, and distilling a filtrate under reduced pressure to remove a solvent; adding acid to regulate pH value, cooling, crystallizing, filtering, collecting precipitated solid and drying to obtain pure product of stable isotope labeled methotrexate.
The reaction of this step is as follows:
wherein the stable isotope Labeled Methotrexate has a chemical structure shown in a formula (Labeled-methotrexa), and X is 12 C or 13 C, Y is H or D.
In the step (1), the organic solvent is any one or more of tetrahydrofuran, dioxane, methyl tertiary butyl ether, diethyl ether, acetone, toluene, xylene, 2-methyltetrahydrofuran, N-dimethylformamide, N-dimethylacetamide, methanol, ethanol, isopropanol, N-propanol, benzene and chlorobenzene; the alkali is any one or more of lithium carbonate, sodium carbonate, potassium carbonate, cesium carbonate, sodium bicarbonate, potassium bicarbonate, sodium hydride, potassium hydride, sodium tert-butoxide, potassium tert-butoxide, sodium methoxide, sodium ethoxide and the like and aqueous solutions thereof; the phase transfer catalyst is tetrabutylammonium chloride, tetrabutylammonium bromide, benzyltriethylammonium chloride, tetrabutylammonium bisulfate, trioctylmethylammonium chloride, dodecyltrimethylammonium chloride, tetradecyltrimethylammonium chloride, 18 crown 6, 15. Crown 5, cyclodextrin, polyethylene glycol; stable isotope labeled methyl p-toluenesulfonate is deuterium labeled, 13 C-tag or 13 C and deuterium double-labeled methyl p-toluenesulfonate; the reaction temperature is 30-120 ℃; the reaction time is 12-48 hours.
Preferably, the organic solvent in the step (1) is N, N-dimethylformamide, tetrahydrofuran or 2-methyltetrahydrofuran;
more preferably, the organic solvent is N, N-dimethylformamide.
Preferably, the base in the step (1) is sodium carbonate, potassium carbonate, cesium carbonate, sodium bicarbonate, potassium bicarbonate or sodium tert-butoxide, potassium tert-butoxide; the molar ratio of the compound I to the alkali is (1:1) - (1:3);
more preferably, the base is potassium carbonate.
Preferably, the phase transfer catalyst in the step (1) is tetrabutylammonium bromide, benzyl triethylammonium chloride, trioctylmethyl ammonium chloride, dodecyl trimethyl ammonium chloride; the molar ratio of the compound I to the phase transfer catalyst is (1:2) - (1:10);
more preferably, the phase transfer catalyst is tetrabutylammonium bromide.
Preferably, the molar ratio of the compound (I) to the diethyl N- (4-aminobenzoyl) -L-glutamate is (1:1-1:1.5).
Preferably, the reaction temperature in the step (1) is 50 to 90 ℃.
Preferably, the reaction time in the step (1) is 18 to 24 hours.
In the step (2), the organic solvent is any one or more of N, N-dimethylformamide, N-dimethylacetamide, dimethyl sulfoxide, acetonitrile, tetrahydrofuran and dioxane; the acid binding agent is any one or more of pyridine, triethylamine, diisopropylethylamine, lithium carbonate, sodium carbonate, potassium carbonate, cesium carbonate, calcium carbonate, sodium bicarbonate, potassium bicarbonate and 1, 8-diazabicyclo undec-7-ene; the reaction temperature is 20-80 ℃; the reaction time is 8-36 hours; the molar ratio of the compound (II) to the acid binding agent is (1:2) - (1:10).
Preferably, the organic solvent in the step (2) is N, N-dimethylformamide, N-dimethylacetamide or dimethylsulfoxide;
more preferably, the organic solvent is N, N-dimethylacetamide.
Preferably, the acid-binding agent in the step (2) is pyridine, triethylamine, diisopropylethylamine, 1, 8-diazabicyclo undec-7-ene;
more preferably, the acid binding agent is diisopropylethylamine.
Preferably, the reaction temperature in the step (2) is 40 to 60 ℃.
Preferably, the reaction time in the step (2) is 12 to 18 hours.
In the step (3), the organic solvent is any one or more of methanol, ethanol, n-propanol and isopropanol; the inorganic base is any one or more of lithium hydroxide, sodium hydroxide and potassium hydroxide; the acid used for regulating the pH is any one or more of hydrochloric acid, sulfuric acid, formic acid, acetic acid, trifluoroacetic acid and hydrobromic acid; the reaction temperature is between 10 ℃ below zero and 50 ℃; the reaction time is 1 to 8 hours; the molar ratio of the compound (III) to the inorganic base is (1:2) to (1:5).
Preferably, the organic solvent in the step (3) is methanol or ethanol;
more preferably, the organic solvent is ethanol;
preferably, the inorganic base in the step (3) is sodium hydroxide;
preferably, the acid used for adjusting the pH in the step (3) is hydrochloric acid;
preferably, the reaction temperature in the step (3) is 20-40 ℃;
preferably, the reaction time in the step (3) is 4 to 6 hours.
Compared with the prior art, the invention has the beneficial effects that: the preparation method of the stable isotope labeled methotrexate internal standard reagent for monitoring the blood concentration of the clinical treatment drug has the advantages of simple and easily obtained raw materials, low cost, no use of a highly toxic methylation reagent, short synthesis step, mild reaction conditions, few byproducts of a final product, easy separation and purification, high chemical purity, high total yield, undiluted isotope abundance and the like. The preparation route and the method of the stable isotope labeled methotrexate internal standard reagent have not been reported in the literature, and the chemical purity of the stable isotope labeled methotrexate prepared by the method is more than 99%, and the isotope abundance is more than 99%. The stable isotope labeled methotrexate internal standard reagent for monitoring the blood concentration of the clinical therapeutic drug prepared by the preparation method has important significance for the development of relevant clinical therapeutic drug blood concentration monitoring technology in China.
Description of the drawings:
fig. 1: n- (4-methylaminobenzoyl) -L-glutamic acid diethyl ester (N-methyl-D) 3 ) A kind of electronic device 1 H NMR chart
Fig. 2: n- (4-methylaminobenzoyl) -L-glutamic acid diethyl ester (N-methyl-D) 3 ) LC-MS diagram of (c)
Fig. 3: n- [4- [ [ (2, 4-diamino-6-pteridine) methyl]Methylamino group]Benzoyl compounds]-L-glutamic acid diethyl ester (N-methyl-D) 3 ) A kind of electronic device 1 H NMR chart
Fig. 4: n- [4- [ [ (2, 4-diamino-6-pteridine) methyl]Methylamino group]Benzoyl compounds]-L-glutamic acid diethyl ester (N-methyl-D) 3 ) LC-MS diagram of (c)
Fig. 5: methotrexate (N-methyl-D) 3 ) A kind of electronic device 1 H NMR chart
Fig. 6: methotrexate (N-methyl-D) 3 ) LC-MS diagram of (c)
Fig. 7: methotrexate (N-methyl-D) 3 ) HPLC diagram of (2)
Detailed Description
The invention is further described below in connection with the examples and the drawings thereof, but is not intended to limit the scope of the invention in any way.
Example 1: n- (4-methylaminobenzoyl) -L-glutamic acid diethyl ester (N-methyl-D) 3 ) Preparation
In a 100mL three-necked flask, methyl deuterated methylbenzenesulfonate (1.42 g,7.5 mmol), diethyl N- (4-aminobenzoyl) -L-glutamate (2.74 g,8.25 mmol), tetrabutylammonium bromide (12.46 g,37.5 mmol), 20% aqueous potassium carbonate solution (10 mL), N-dimethylformamide (30 mL) were added; reacting at 70 ℃ for 20 hours; TLC was used to follow the completion of the reaction of the starting materials, the reaction mixture was cooled to room temperature, 200mL of water was added, extracted with ethyl acetate (3X 100 mL), the organic phases were combined, washed successively with saturated sodium bicarbonate solution, saturated sodium chloride solution, dried over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure, and the residue was purified by column chromatography to give stable isotope-labeled N- (4-methylaminobenzoyl) -L-glutamic acid diethyl ester (N-methyl-D) 3 ) (1.9 g, 75% yield). 1 H NMR(DMSO-D 6 600 MHz): delta 8.22 (d, j=6 hz, 1H), 7.69 (d, j=12 hz, 2H), 6.54 (d, j=12 hz, 2H), 6.20 (s, 1H), 4.40-4.36 (m, 1H), 4.12-4.08 (m, 2H), 4.06 (dd, j=24, 6hz, 2H), 2.42 (t, j=6 hz, 2H), 2.11-2.05 (m, 1H), 2.01-1.95 (m, 1H), 1.20-1.16 (m, 6H) see fig. 1; LC-MS (ESI+) 340[ M+H ]] + See fig. 2.
Example 2: n- [4- [ [ (2, 4-diamino-6-pteridine) methyl]Methylamino group]Benzoyl compounds]-L-glutamic acid diethyl ester (N-methyl-D) 3 ) Is prepared from
Into a 100mL three-necked flask, stable isotope labeled N- (4-methylaminobenzoyl) -L-glutamic acid diethyl ester (N-methyl-D) was added 3 ) (0.63 g,1.84 mmol), 6- (bromomethyl) -2, 4-pteridine diamine hydrobromide (0.69 g,1.84 mmol), N-dimethylacetamide (20 mL), diisopropylethylamine (0.71 g,5.52 mmol); reacting for 16 hours at 50 ℃; TLC was followed by complete reaction of the starting materials, the solvent was distilled off under reduced pressure, 20mL of water was added, extraction was performed with ethyl acetate (3X 30 mL), the organic phases were combined, washed successively with saturated sodium bicarbonate solution, saturated sodium chloride solution, dried over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure, and the residue was taken outSubjecting the obtained product to column chromatography to obtain N- [4- [ [ (2, 4-diamino-6-pteridine) methyl group]Methylamino group]Benzoyl]-L-glutamic acid diethyl ester (N-methyl-D) 3 ) (0.77 g, 82% yield). 1 HNMR(DMSO-D 6 600 MHz): delta 8.58 (s, 1H), 8.30 (d, j=6 hz, 1H), 7.73 (d, j=6 hz, 2H), 7.67 (brs, 1H), 7.44 (brs, 1H), 6.83 (d, j=6 hz, 1H), 6.62 (brs, 1H), 4.78 (s, 2H), 4.40-4.36 (m, 1H), 4.11-4.07 (m, 2H), 4.05 (dd, j=12, 6hz, 2H), 2.41 (t, j=12 hz, 2H), 2.10-2.04 (m, 1H), 2.00-1.94 (m, 1H), 1.19-1.14 (m, 6H) see fig. 3; LC-MS (ESI+) 514[ M+H ]] + See fig. 4.
Example 3: methotrexate (N-methyl-D) 3 ) Is prepared from
In a 50mL three-necked flask, N- [4- [ [ (2, 4-diamino-6-pteridine) methyl group was added]Methylamino group]Benzoyl compounds]-L-glutamic acid diethyl ester (N-methyl-D) 3 ) (0.17 g,0.33 mmol), methanol (5 mL), sodium hydroxide (0.04 g,1 mmol); and reacting at 30 ℃ for 5 hours; after TLC tracking reaction raw materials are reacted completely, decompressing and distilling to remove the solvent, adding 10mL of water, adjusting the pH value to 7 by using 1mol/L hydrochloric acid, standing and refrigerating, and crystallizing; the yellow crystals precipitated were collected by filtration and dried under vacuum to give stabilized homoleptin-labeled methotrexate (N-methyl-D) 3 ) (0.12 g, yield 80%). 1 H NMR(DMSO-D 6 600 MHz): 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 to 4.33 (m, 1H), 2.32 (t, j=6hz, 2H), 2.08 to 2.02 (m, 1H), 1.95 to 1.88 (m, 1H), see fig. 5; LC-MS (ESI+) 458[ M+H ]] + See fig. 6; HPLC measured chemical purity = 99.31%, see figure 7. Isotopic abundance=99.64%, see table 1.
TABLE 1 methotrexate (N-methyl-D) 3 ) Isotope abundance of (2)
The quality standard meets the quality requirement of the stable isotope labeled internal standard reagent required by the liquid chromatography-tandem mass spectrometry for monitoring the blood concentration of the clinical therapeutic drug.
13 C label 13 CD 3 The synthetic route process of the double-labeled stable isotope labeled methotrexate is the same as that of the stable homoleptic labeled methotrexate in the above embodiment.
The foregoing is illustrative of a preferred embodiment of the present invention, but the present invention should not be limited to the disclosure of this embodiment. So that equivalents and modifications will fall within the scope of the invention, all within the spirit and scope of the invention as disclosed.
Claims (7)
1. The chemical structure of the stable isotope labeled methotrexate internal standard reagent is shown as a formula (1):
wherein XY 3 Is a CD 3 The preparation method is characterized by comprising the following steps of:
wherein XY is 3 Is a CD 3 The preparation method comprises the following steps:
(1) Adding stable isotope labeled methyl p-toluenesulfonate in the presence of an organic solvent, alkali and a phase transfer catalyst, then adding N- (4-aminobenzoyl) -L-diethyl glutamate, and reacting for 12-48 hours at 30-120 ℃; after TLC tracking reaction raw materials are reacted completely, quenching a reaction mixed solution by water, filtering, extracting a filtrate by using an organic solvent, combining organic phases, washing by using a saturated sodium bicarbonate solution and a saturated sodium chloride solution in sequence, adding a drying agent for drying, distilling under reduced pressure to remove the solvent, and carrying out column chromatography on residues to obtain stable isotope labeled N- (4-methylaminobenzoyl) -L-diethyl glutamate;
the reaction of this step is as follows:
wherein the chemical structure of the stable isotope labeled methyl tosylate is shown as a formula (I), the chemical structure of the stable isotope labeled diethyl N- (4-methylaminobenzoyl) -L-glutamate is shown as a formula (II), and the XY is shown as a formula (II) 3 Is a CD 3 ；
(2) Dissolving stable isotope labeled N- (4-methylaminobenzoyl) -L-glutamic acid diethyl ester in an organic solvent, adding 6- (bromomethyl) -2, 4-pteridine diamine hydrobromide and an acid binding agent, and reacting for 12-48 hours at 20-80 ℃; after TLC tracking reaction raw materials are reacted completely, quenching a reaction mixed solution by water, filtering, extracting a filtrate by using an organic solvent, combining organic phases, washing by using a saturated sodium bicarbonate solution and a saturated sodium chloride solution in sequence, adding a drying agent for drying, distilling under reduced pressure to remove the solvent, and obtaining stable isotope labeled N- [4- [ [ (2, 4-diamino-6-pteridine) methyl ] methylamino ] benzoyl ] -L-diethyl glutamate from residues by column chromatography;
the reaction of this step is as follows:
wherein the stable isotope labels N- [4- [ [ (2, 4-diamino-6-pteridine) methyl]Methylamino group]Benzoyl compounds]The chemical structure of the-L-diethyl glutamate is shown as a formula (III), wherein XY is shown as the formula (III) 3 Is a CD 3 ；
(3) Dissolving stable isotope labeled N- [4- [ [ (2, 4-diamino-6-pteridine) methyl ] methylamino ] benzoyl ] -L-glutamic acid diethyl ester in an organic solvent, adding inorganic base, and reacting for 2-12 hours at 20-100 ℃; after TLC tracking reaction raw materials are reacted completely, filtering a reaction solution, and distilling a filtrate under reduced pressure to remove a solvent; adding acid to adjust the pH value, cooling for crystallization, filtering, collecting precipitated solid, and drying to obtain pure product stable isotope labeled methotrexate;
the reaction of this step is as follows:
wherein the chemical structure of the stable isotope labeled methotrexate is shown as a formula (1), wherein XY 3 Is a CD 3 。
2. The method for preparing the stable isotope labeled methotrexate internal standard reagent according to claim 1, wherein the method comprises the following steps: in the step (1), the organic solvent is any one or more of tetrahydrofuran, dioxane, methyl tertiary butyl ether, diethyl ether, acetone, toluene, xylene, 2-methyltetrahydrofuran, N-dimethylformamide, N-dimethylacetamide, methanol, ethanol, isopropanol, N-propanol, benzene and chlorobenzene;
the alkali is any one or more of lithium carbonate, sodium carbonate, potassium carbonate, cesium carbonate, sodium bicarbonate, potassium bicarbonate, sodium hydride, potassium hydride, sodium tert-butoxide, potassium tert-butoxide, sodium methoxide, sodium ethoxide and aqueous solution thereof;
the phase transfer catalyst is any one or more of tetrabutylammonium chloride, tetrabutylammonium bromide, benzyl triethylammonium chloride, tetrabutylammonium bisulfate, trioctylmethyl ammonium chloride, dodecyl trimethyl ammonium chloride, tetradecyl trimethyl ammonium chloride, 18-crown-6, 15-crown 5, cyclodextrin and polyethylene glycol;
the reaction temperature is 30-120 ℃; the reaction time is 12-48 hours.
3. The method for preparing the stable isotope labeled methotrexate internal standard reagent according to any one of claims 1 or 2, wherein the method comprises the following steps: in the step (1), the molar ratio of the compound (I) to the alkali is (1:1) - (1:3); the molar ratio of the compound (I) to the phase transfer catalyst is (1:2) to (1:10); the molar ratio of the compound (I) to the N- (4-aminobenzoyl) -L-diethyl glutamate is (1:1-1:1.5); the reaction temperature is 50-90 ℃; the reaction time is 18-24 hours.
4. The method for preparing the stable isotope labeled methotrexate internal standard reagent according to claim 1, wherein the method comprises the following steps: in the step (2), the organic solvent is any one or more of N, N-dimethylformamide, N-dimethylacetamide, dimethyl sulfoxide, acetonitrile, tetrahydrofuran and dioxane;
the acid binding agent is any one or more of pyridine, triethylamine, diisopropylethylamine, lithium carbonate, sodium carbonate, potassium carbonate, cesium carbonate, calcium carbonate, sodium bicarbonate, potassium bicarbonate and 1, 8-diazabicyclo undec-7-ene;
the reaction temperature is 20-80 ℃; the reaction time is 8-36 hours.
5. The method for preparing the stable isotope labeled methotrexate internal standard reagent according to any one of claims 1 or 4, wherein the method comprises the following steps: in the step (2), the molar ratio of the compound (II) to the acid-binding agent is (1:2) - (1:10); the reaction temperature is 40-60 ℃; the reaction time is 12-18 hours.
6. The method for preparing the stable isotope labeled methotrexate internal standard reagent according to claim 1, wherein the method comprises the following steps: in the step (3), the organic solvent is any one or more of methanol, ethanol, n-propanol and isopropanol; the inorganic base is any one or more of lithium hydroxide, sodium hydroxide and potassium hydroxide; the acid used for regulating the pH is any one or more of hydrochloric acid, sulfuric acid, formic acid, acetic acid, trifluoroacetic acid and hydrobromic acid; the reaction temperature is between 20 ℃ below zero and 100 ℃; the reaction time is 2-12 hours.
7. The method for preparing the stable isotope labeled methotrexate internal standard reagent according to any one of claims 1 or 6, wherein the method comprises the following steps: in the step (3), the reaction temperature is 20-40 ℃; the reaction time is 4 to 6 hours.
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