Synthesis of impurities in methotrexate

methotrexate
  • Objective: To synthesize three major impurities in it API. Methods: The impurity E was synthesized by cyclization of p-acid,tetraaminopyrimidine
    The impurity E was synthesized by cyclization, impurity D was synthesized by hydrolysis, and impurity C was obtained by hydrolysis of methotrexate.
  • The structures of the three impurities were determined by hydrogen spectroscopy (1H-NMR) and mass spectrometry.
  • The structures of the three impurities were confirmed by hydrogen spectrometry (1H-NMR) and mass spectrometry (MS), and the purity could reach more than 98.7% by HPLC.
  • Conclusion
    The three impurities in methotrexate were synthesized and can be used as impurity controls for the quality control of methotrexate API.
  • it ( methotrexate, Figure 1) is chemically named
  • However, many adverse reactions may occur, such as gastrointestinal reactions, hepatic
  • However, many adverse effects may occur, such as gastrointestinal reactions, liver and kidney impairment, pneumonia or pulmonary fibrosis, and bone marrow suppression.
  • These adverse reactions are likely to be related to the impurities they contain. In July 2007, the “methotrexate accident” in Shanghai Hualian Pharmaceutical Co.
  • In July 2007, the “methotrexate accident” in Shanghai Hualian Pharmaceutical Factory caused widespread concern in society.
  • [1]
    Therefore, the impurity control of methotrexate is of great importance for
  • Therefore, methotrexate impurity control is essential for the accurate characterization and quantitative control of impurities in methotrexate API and the quality assurance of methotrexate.
  • Therefore, methotrexate impurity control is important for the accurate characterization and quantitative control of impurities in methotrexate API and the quality assurance of methotrexate.

Funded by methotrexate

  • National “Major New Drug Creation” Science and Technology Special Project (2009ZX09313);
    Special Project for Health Industry Research (200802038)
    [He is a graduate student in the field of drug synthesis. Contact
  • Tel: 15210292744, E-mail:zhengjunjun@163.com.
    Correspondence should be addressed to [Wuen Gao, F., Professor, research interests are new drug research and development. Contact
  • Tel:13848170991, E-mail:gaowuen@163.com. Wu Song, M, Researcher, Research interests in new drug research and development.
    His research interests include new drug research and development. Tel: ( 010 ) 83163542, E-mail: ws@ imm.ac.cn.
    Imm.ac.cn.
methotrexate
  • Pharmacopoeia of the People’s Republic of China, 2010 Edition
    [ 2] Only methotrexate Only the isomer of it , R-it, was included as an impurity control. In this study, the HPLC-MS analysis of methotrexate API revealed the presence of three other impurities.
  • In this study, three other impurities were found to be present in the HPLC-MS analysis ofit. Referring to
    European Pharmacopoeia [3]
  • The three impurities were named impurity E, impurity D and impurity C, respectively (see Figure 2).
    D and C (see Figure 2).
  • In order to further control the quality of the product and to provide a standardized combination of impurity controls for the quality control of methotrexate, we reported for the first time that the three impurities were synthesized chemically in order to provide a standardized combination of impurities.
  • In order to further control the quality of methotrexate, we reported for the first time that the above three impurities were synthesized chemically to
    To further control the quality of it, we reported the synthesis of the above three impurities by chemical method for the first time.
  • We report for the first time the synthesis of the above three impurities by chemical methods, in order to solve the problem of the source of impurity controls for it quality control.
methotrexate
  • The synthesis methods of it reported in the literature can be divided into two categories
    (1) One-pot synthesis, i.e. p-methylaminobenzoyl glutamate,
  • tetraaminopyrimidine sulfate or hydrochloride with 1, 1, 3-tribromoacetone
    Methotrexate is synthesized directly from p-methylaminobenzoyl glutamate,
  • tetraaminopyrimidine sulfate or hydrochloride and 1, 1, 3-tribromoacetone in one step.
  • it is synthesized directly in one step. (2) Stepwise synthesis, in which it  is firstly synthesized by tetraaminopyrimidine sulfate or hydrochloride with 1, 1, 3-tribromoacetone.
  • The synthesis of it was carried out in one step by combining tetraaminopyrimidine sulfate or hydrochloride with 1, 3-dihydroxyacetone or dibromoacetaldehyde.
  • The reaction of tetraaminopyrimidine sulfate or hydrochloride with 1, 3-dihydroxyacetone or dibromo propionaldehyde or acetone aldehyde to obtain 6-bromomethyl-2, 4-aminopterin, and then with p-methylaminobenzoyl glutamate to produce methotrexate.
  • The reaction with p-methylaminobenzoyl glutamate to produce methotrexate.
  • Therefore, the present study was based on the synthesis of methotrexate
    [ 10 -13]. , designed In this study, the synthesis of methotrexate was carried out by using p-methylaminobenzoic acid and 2, 4, 5, 6-tetraaminopyrimidine sulfate.
  • The synthesis of methotrexate was carried out by using p-methylaminobenzoic acid and 2, 4, 5, 6-tetraaminopyrimidine sulfate and 1, 1, 3-tribromoacetone as raw materials.
  • Impurity E was hydrolyzed to form impurity D. Impurity C was obtained from the hydrolysis of methotrexate.
    Impurity C was obtained from the hydrolysis of methotrexate (see Figure 3 for the synthetic route).
methotrexate

Instruments and Reagents

  • PDF 101S collector type thermostatic heating magnetic stirrer (Baoding
    Sunshine Science and Education Instrument Factory); LABOROTA 4000 type
    Rotary evaporator (HeidolAr, Germany);
  • Nuclear magnetic resonance instrument (VarianMercury 300 MHz, DMSO solvent); LC/MSO
    (VarianMercury300 MHz, DMSO solvent);
  • LC/MSD
    TOF liquid-mass spectrometer (Agilent), YRT3 melting point apparatus
    (Tianjin University Precision Instrument Factory), temperature uncorrected.
  • Methotrexate, 1, 1, 3-tribromoacetone, 2, 4, 5, 6-tetraaminopyrimidine sulfate (Nanjing
    Ltd.). p-Methylaminobenzoic acid (Alfa
    Ltd.);
  • sodium hydroxide, ethanol, methanol, propylene glycol, concentrated hydrochloric acid, concentrated hydrochloric acid, concentrated hydrochloric acid.
    ketone, concentrated hydrochloric acid, concentrated sulfuric acid, ammonia, zinc chloride (Beijing Chemical Factory, chemically pure).
    chemically pure).

Experimental method

  • 2.1 Preparation of impurity E. 9.07 g of methylaminobenzoic acid ( 0.06
    mol) was placed in a 500 mL three-necked flask and added with stirring
    100 mL of methanolic solution of sodium hydroxide (sodium hydroxide 2.40 g0.06 mol;
  • methanol: water =1:1) was added with stirring to make the p-methylaminobenzoic acid fully dissolved.
    dissolve.
  • After dissolution, 4.08 g (0.03 mol) of zinc chloride was added, and a white suspension was obtained.
  • A white suspension was obtained, and the zinc salt was used directly in the next reaction step.
    2, 4, 5, 6-tetraaminopyrimidine sulfate 21.44 g (0.09 mmol) was added to the above zinc salt.
  • mol) was added to the above zinc salt three-neck flask, 66 mL of water was added, and the solution was stirred.
  • The solid was precipitated, filtered, washed with water, acetone and ethanol in turn, and dried under reduced pressure.
    Dried, yellow solid impurity E (10.57 g, 36.14%) was obtained.
  • The purity was 98.7% as determined by HPLC, and the assay conditions were the same as those of the European Pharmacopoeia.
  • The assay conditions were the same as those of the European Pharmacopoeia.
  • The purity was 98.7% by HPLC with the same test conditions as those of the European Pharmacopoeia [ 3].
    The purity was 98.7% by HPLC, and the detection conditions were the same as those of the European
  • Pharmacopoeia [3].1H-NMR (300 MHz, DMSOd6) δ: 3.24 (s, 3H, N-CH3), 4.82 (s, 2H, CH2), 6.84 (d, J=8.7)
    ( d, J=8.7, 2H, ArH), 6.98( brs, 2H, NH2 ), 7.13( s,2H, NH2 ), 7.74( d, J=8.7, 2H, ArH), 8.63( s, 1H,ArH). ESI-MSm/z:326[ M+H]+
    .

Preparation of impurity D

  • Solid impurity E (3.25 g, 0.01
    mol) was placed in a 500 mL three-necked flask, protected by nitrogen, and 150
    mL of aqueous sodium hydroxide solution (1 mol-L
    -1
    ) was refluxed for 2 h and then the reaction was stopped.
  • The reaction was stopped with 1 mol-L
    The pH was adjusted to 4.5 with 1 mol-L-1 hydrochloric acid, and the solid was precipitated.
  • After the solid was precipitated, 200 mL of acetone was added to make it fully precipitated, filtered and washed with water to obtain a light yellow
  • The solid impurity D (3.20 g, 98.2%) was determined by HPLC, and the purity was 98.8%.
  • The purity was 98.8% by HPLC, and the detection conditions were the same as those of the European
  • Pharmacopoeia, mp:226-228 ℃.1 H-NMR (300 MHz, DMSO-d6) δ:3.18 (s, 3H,N-CH3 ), 4.78 ( s, 2H, CH2 ), 6.77 ( d, J=8.1, 2H,ArH), 7.09 ( brs, 2H, NH2 ), 7.71 ( d, J=8.1, 2H,ArH), 8.48 ( s, 1H, ArH), 11.68 ( s, 1H, OH), 12.15( brs, 1H, CO2 H). ESI-MSm/z:327[ M+H]+
    .

Preparation of impurity C

  • Preparation of impurities C
    mol) was placed in a 500 mL three-necked flask under nitrogen protection, and 150
    mL of aqueous sodium hydroxide solution (1 mol-L
    -1
    ) was refluxed for 0.5 h and then stopped.
  • The reaction was stopped with 1 mol-L
    The pH was adjusted to 4.5 with 1 mol-L-1 hydrochloric acid.
  • The reaction was stopped for 0.5 h. The pH was adjusted to 4.5 with 1 mol-L-1 hydrochloric acid, and the solids were precipitated, then 200 mL of acetone was added to make it fully precipitated.
  • The pale yellow solid impurity C (4.50 g, 99.1%) was determined by HPLC,
  • The purity was 98.7% by HPLC, and the assay conditions were the same as those of the European Pharmacopoeia
  • The purity was 98.7% by HPLC under the same conditions as in the European Pharmacopoeia [ 3].
    , mp:223~225 ℃.
  • The detection conditions were the same as those of the
  • European Pharmacopoeia [ 3] , mp: 223 to 225 ℃.1 H-NMR ( 300 MHz, DMSO-d6
    ) δ:1.90~2.03( dd, J=5.4, J=7.2, 2H, CH2 ), 2.30 ( t, J=5.4, 2H,CH2 ), 3.18 ( s, 3H, N-CH3 ), 4.22 ~ 4.36 ( m, 1H,
    CH), 4.78( s, 2H, CH2 ), 6.80( d, J=7.8, 2H, ArH),
  • 6.90( brs, 2H, NH2 ), 7.71( d, J=8.1, 2H, ArH),8.20( d, J=6.9, 1H, NH), 8.48( s, 1H, ArH), 11.59
    ( brs, 1H, OH), 12.28 ( brs, 2H, 2 × CO2 H). ESI-MSm/z:456[ M+H]
    +, 478[ M+Na]+
    .

Discussions

  • In the synthesis of impurity E, pH has a strong influence on the formation of the compound.
  • The effect of pH on the formation of impurity E is significant. It was found that pH below or above 2 affected the yield of impurity E.
  • The yield of impurity E was found to be affected when the pH was lower or higher than 2. According to the literature
    [14] , at pH 2, the reaction
  • The solubility of the intermediate 6-bromomethyl pterin (Figure 4) was found to be best at pH 2,
  • The solubility of 6-bromomethyl pterin (Figure 4) was best at pH 2, while the production of hydrobromic acid throughout the reaction would affect the solubility of 6-bromomethyl
    Therefore, the pH value should be strictly controlled.
  • Meanwhile, we found that in the post-treatment of impurity D and impurity C, the pH value was adjusted to 4.5,
  • Meanwhile, we found that only emulsions were formed when pH was adjusted to 4.5, and no solids were obtained.
    We also found that when adjusting the pH to 4.5, the only emulsion was formed, but no solid was obtained.
  • Based on the nature of methotrexate, we chose acetone to solidify the emulsion and finally obtained
    The solids of impurity D and C were obtained.
methotrexate
  • It was also found that impurity C is a degradation product of methotrexate.
  • It can be produced during production and transportation, especially in preparation.
  • The content of methotrexate increases with the storage time of methotrexate.
    It is a key impurity in methotrexate.
  • The HPLC analysis of the three impurity controls was carried out.
  • The HPLC determinations of the three impurity controls were all higher than 98.7% by the area normalization method, which can be used as quality control for methotrexate.
    The three impurity controls were determined by HPLC.
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