Preparation method and application of methotrexate intermediate

Abstract

The invention relates to a preparation method and application of a methotrexate intermediate, and particularly provides a synthetic route for synthesizing p-methylaminobenzoyl-L-glutamic acid sodium salt, the route uses p-aminobenzoyl-L-glutamic acid (SM1) with easily available raw materials as a starting material, the starting material and paraformaldehyde are subjected to a reductive amination reaction under the action of sodium methoxide and sodium borohydride, and the methotrexate intermediate is obtained. And reacting to generate the p-methylaminobenzoyl-L-glutamic acid sodium salt (M1). According to the route, the reaction synthesis conditions are simplified, the reaction steps are shortened, the generation of byproducts is reduced, and the purity and the yield of the target product p-methylaminobenzoyl-L-sodium glutamate are improved.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a preparation method of a methotrexate intermediate and an application thereof. Background Art

[0002] Methotrexate is an anti-folate anti-tumor drug that inhibits the synthesis of tumor cells by inhibiting dihydrofolate reductase, thereby inhibiting the growth and reproduction of tumor cells. The synthesis of methotrexate is obtained by condensing the pterin nucleus and the side chain (M2) (e.g. Figure 1 As shown), the synthesis process of the side chain p-methylaminobenzoyl-L-glutamic acid zinc salt is relatively long and the operation is complicated.

[0003] According to the literature reports, there are mainly the following methods for synthesizing the methotrexate intermediate p-methylaminobenzoyl-L-glutamic acid: 1. Figure 2 As shown in a), document WO9920626 uses p-methylaminobenzamide azide as a starting material and reacts with L-glutamic acid to produce p-methylaminobenzoyl-L-glutamic acid. Figure 2 As shown in b), documents CN113735735 and CN113461689 use chlorobenzoyl-L-glutamic acid and methylamine as raw materials, use highly active copper catalysts, and cooperate with fixed bed reaction systems to generate p-methylaminobenzoyl-L-glutamic acid under high pressure catalysis. 3. Figure 2 As shown in c), the document US3892801 uses p-nitrobenzoyl chloride as a starting material to react with glutamic acid to generate p-nitrobenzoyl-L-glutamic acid, which is reduced by zinc powder to obtain p-aminobenzoyl-L-glutamate zinc, which is then nitrosated and methylated to obtain the target product. 4. Figure 3 As shown in a), the document US4211883, J Am Chem Soc, 1948.70.1922 uses p-aminobenzoic acid as the starting material, which is first reacted with formic acid and then with thionyl chloride to obtain 2, 2 which is then reacted with glutamic acid to obtain 3, 3 which is then alkaline hydrolyzed to obtain the target product. 5. Figure 3 b), Chinese Pharmaceutical Industry Impurities, "Methotrexate Side Chain Synthesis Process", 1992, 23 (2), 49-51. Based on route 3 and route 4, some improvements were made, using cheap and readily available p-nitrobenzoic acid as the starting material, reducing p-aminobenzoic acid, reacting with dimethyl sulfate to obtain p-aminobenzoic acid, and then reflux reacting with formic acid to obtain 4-(N-formyl-N-methyl)aminobenzoic acid, acylating with dichlorothionyl to obtain 4-(N-formyl-N-methyl)aminobenzoyl chloride, reacting with L-glutamic acid, and then hydrolyzing and adjusting the acid to obtain the target product p-aminobenzoyl-L-glutamic acid. 6. Figure 3c), the document "High-efficiency synthesis of ethyl 4-[N-methyl-N-(2,3-aziridine)amino]benzoate and diethyl N-{4-[N-methyl-N-(2,3-aziridine)amino]benzoyl}-L-glutamate", Chinese Pharmacy (2010), 19(2), 141-145. Diethyl 4-aminobenzoyl-L-glutamate and methyl iodide are reacted to generate diethyl p-methylaminobenzoyl-L-glutamate.

[0004] Among the above synthetic routes of methotrexate intermediates, the starting material of route 1, p-aminobenzamide azide, is difficult to purchase and unstable, posing safety risks. Route 2 uses a highly active copper-based catalyst, a fixed bed reaction system, and a high-pressure catalytic reaction, which is highly dangerous and difficult to industrialize. The starting material of route 3 is easy to obtain and cheap, but the synthetic route is long, the process operation is cumbersome, and the total yield is low by 10 to 20%. The starting material of route 4 is difficult to obtain and difficult to purchase. Route 5 is improved on the basis of routes 3 and 4, using dimethyl sulfate to methylate, which also has the disadvantages of a long synthetic route, cumbersome process operation, and a low total yield of 20 to 30%. Route 6 uses methyl iodide to methylate, and dimethyl substances are easy to obtain, which are difficult to remove and have a low overall yield (23-64%).

[0005] In summary, there are many methods for synthesizing methotrexate intermediates, but they all have certain problems, such as difficulty in obtaining raw materials, high requirements for reaction conditions, long routes, and low yields. Summary of the invention

[0006] In order to solve the above problems, the inventors of the present application have provided a new synthetic route for synthesizing p-aminobenzoyl-L-glutamate zinc salt after in-depth research and exploration. The route uses p-aminobenzoyl-L-glutamate (SM1) which is easily available as a starting material, and undergoes a reduction amination reaction with paraformaldehyde under the action of sodium methoxide and sodium borohydride to generate p-aminobenzoyl-L-glutamate sodium salt (M1). After that, the sodium salt is acidified and ion-exchanged to obtain p-aminobenzoyl-L-glutamate zinc salt (M2). The inventors found that in the route, the preparation of p-aminobenzoyl-L-glutamate sodium salt (M1) is the most critical step.

[0007] In the method for preparing p-aminobenzoyl-L-glutamate sodium salt (M1) provided by the present invention, p-aminobenzoyl-L-glutamate and paraformaldehyde are first subjected to an imidization reaction under the action of sodium methoxide to generate an intermediate int1, and int1 does not need to be separated and purified (i.e., it is a one-pot reaction), and p-aminobenzoyl-L-glutamate sodium salt (M1) can be obtained by directly reducing it with sodium borohydride.

[0008]

[0009] After a detailed study of the route, the inventor found that the main impurity in the product is the starting material p-aminobenzoyl-L-glutamic acid (SM1). If the starting material SM1 residue is large (>2%), the subsequent use of M1 to further prepare methotrexate will produce the corresponding derivative impurity B (>0.3%), and the impurity is difficult to remove by post-treatment and purification, resulting in unqualified methotrexate products. Therefore, in order to be able to prepare methotrexate that meets the pharmacopoeia quality standards (impurity K <0.05%, impurity B <0.3%), the following standards need to be met when preparing M1: the starting material impurity SM1 needs to be controlled within 2%.

[0010]

[0011] In order to prepare the target product M1 that meets the above standards, the inventors further conducted in-depth research on the above route, and finally unexpectedly found that by controlling some reaction conditions (such as the amount of sodium methoxide in the imidization reaction, the reaction temperature and / or the reaction time, etc.), the prepared target product M1 has a purity greater than 98.0%, and the residual p-aminobenzoyl-L-glutamic acid (SM1) is less than 1.0%, which meets the above quality standards. In addition, the reaction system is clear, no solid is precipitated during the reaction, the yield of the target product is high, the steps are simple, and it is conducive to industrial application.

[0012] In summary, the above route simplifies the reaction synthesis conditions, shortens the reaction steps, reduces the generation of by-products, and improves the purity and yield of the target product p-methylaminobenzoyl-L-glutamic acid sodium salt.

[0013] Specifically, in the first aspect of the present invention, the present invention provides a method for preparing a compound represented by formula M1, comprising:

[0014]

[0015] The compound represented by formula SM1 is subjected to a reductive amination reaction with paraformaldehyde in the presence of sodium methoxide and sodium borohydride to obtain a compound represented by formula M1;

[0016] The molar ratio of the sodium methoxide to the compound represented by formula SM1 is 3:1.

[0017] In some embodiments, the method comprises:

[0018] (1-1) allowing the compound represented by formula SM1 to undergo imidization reaction with paraformaldehyde in the presence of sodium methoxide;

[0019] (1-2) subjecting the imidization reaction product of step (1-1) to a reduction reaction with sodium borohydride to obtain a compound represented by formula M1;

[0020] wherein, after the imidization reaction in step (1-1), the reduction reaction in step (1-2) is directly performed without any post-treatment step;

[0021] Preferably, the imidization reaction of step (1-1) and the reduction reaction of step (1-2) are carried out in a one-pot process.

[0022] In some embodiments, the imidization reaction of step (1-1) is carried out for 46-54 h (such as 46 h, 47 h, 48 h, 49 h, 50 h, 51 h, 52 h, 53 h or 54 h, or such as 46-47 h, 47-48 h, 48-49 h, 49-50 h, 50-51 h, 51-52 h, 52-53 h or 53-54 h).

[0023] In some embodiments, the imidization reaction of step (1-1) is carried out at a temperature of 30-40°C (such as 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C or 40°C, or such as 30-31°C, 31-32°C, 32-33°C, 33-34°C, 34-35°C, 35-36°C, 36-37°C, 37-38°C, 38-39°C or 39-40°C).

[0024] In some embodiments, the ratio of the amount of polyformaldehyde to the compound represented by formula SM1 is 1.5:1-6:1 (such as 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1 or 6:1, or such as 1.5:1-2:1, 2:1-2.5:1, 2.5:1-3:1, 3:1-3.5:1, 3.5:1-4:1, 4:1-4.5:1, 4.5:1-5:1, 5:1-5.5:1 or 5.5:1-6:1), preferably 4.5:1.

[0025] In some embodiments, the ratio of the amount of sodium borohydride to the compound represented by formula SM1 is 3:1-5:1 (such as 3:1, 3.2:1, 3.4:1, 3.6:1, 3.8:1, 4:1, 4.2:1, 4.4:1, 4.6:1, 4.8:1 or 5:1, or, such as 3:1-3.2:1, 3.2:1-3.4:1, 3.4:1-3.6:1, 3.6:1-3.8:1, 3.8:1-4:1, 4:1-4.2:1, 4.2:1-4.4:1, 4.4:1-4.6:1, 4.6:1-4.8:1 or 4.8:1-5:1), preferably 3:1.

[0026] In some embodiments, the reduction reaction of step (1-2) is carried out at a temperature of 40-50°C (such as 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C or 50°C, or such as 40-41°C, 41-42°C, 42-43°C, 43-44°C, 44-45°C, 45-46°C, 46-47°C, 47-48°C, 48-49°C or 49-50°C).

[0027] In some embodiments, the reduction reaction of step (1-2) is carried out for 3.5 to 4.5 h (such as 3.5 h, 3.6 h, 3.7 h, 3.8 h, 3.9 h, 4 h, 4.1 h, 4.2 h, 4.3 h, 4.4 h or 4.5 h, or such as 3.5 to 3.6 h, 3.6 to 3.7 h, 3.7 to 3.8 h, 3.8 to 3.9 h, 3.9 to 4 h, 4 to 4.1 h, 4.1 to 4.2 h, 4.2 to 4.3 h, 4.3 to 4.4 h or 4.4 to 4.5 h), preferably 4 h.

[0028] In some embodiments, the solvent for the imidization reaction of step (1-1) and the reduction reaction of step (1-2) is anhydrous methanol.

[0029] In some embodiments, the purity of the sodium methoxide is not less than 96% (such as 96%, 97%, 98%, 99% or 100%, or such as 96%-97%, 97%-98%, 98%-99% or 99%-100%), for example 96%-98%.

[0030] In some embodiments, before the imidization reaction in step (1-1), the compound represented by formula SM1, the paraformaldehyde and the sodium methoxide are pre-mixed by the following method:

[0031] 1) adding the compound represented by formula SM1 into a solvent and stirring to dissolve;

[0032] 2) cooling the solution obtained in step 1) to 0-10°C (such as 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C or 10°C, or such as 0-1°C, 1-2°C, 2-3°C, 3-4°C, 4-5°C, 5-6°C, 6-7°C, 7-8°C, 8-9°C or 9-10°C, preferably 0-4°C), adding the sodium methoxide thereto, and controlling the temperature to be less than 15°C during the addition of the sodium methoxide;

[0033] 3) Adding the paraformaldehyde to the solution obtained in step 2).

[0034] In some embodiments, after the imidization reaction in step (1-1) and before the reduction reaction in step (1-2), the imidization reaction product in step (1-1) and the sodium borohydride are pre-mixed by the following method:

[0035] After the imidization reaction product of step (1-1) is cooled to 0-10°C (such as 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C or 10°C, or such as 0-1°C, 1-2°C, 2-3°C, 3-4°C, 4-5°C, 5-6°C, 6-7°C, 7-8°C, 8-9°C or 9-10°C, preferably 0-4°C), the sodium borohydride is added thereto.

[0036] In some embodiments, after the reduction reaction in step (1-2), the process further comprises:

[0037] (1-3) quenching the reduction reaction;

[0038] (1-4) The mixture obtained in step (1-3) is concentrated under reduced pressure until no solvent is dropped out, to obtain a compound represented by formula M1.

[0039] In some embodiments, quenching the reduction reaction is achieved by the following method: cooling the reduction reaction product of step (1-2) to 0-10°C (such as 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C or 10°C, or such as 0-1°C, 1-2°C, 2-3°C, 3-4°C, 4-5°C, 5-6°C, 6-7°C, 7-8°C, 8-9°C or 9-10°C, preferably 0-4°C), and adding water to quench the reduction reaction.

[0040] In some embodiments, the reduced pressure concentration is carried out at a temperature of 50-60°C (such as 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C or 60°C, or such as 50-51°C, 51-52°C, 52-53°C, 53-54°C, 54-55°C, 55-56°C, 56-57°C, 57-58°C, 58-59°C or 59-60°C).

[0041] In a second aspect of the present invention, the present invention provides a method for preparing methotrexate, comprising:

[0042] (1) Providing a compound represented by formula M1, wherein the compound represented by formula M1 is prepared by the method described in any technical solution of the first aspect;

[0043] (2) acidifying and ion-exchanging the aqueous solution of the compound represented by formula M1 to obtain the compound represented by formula M2;

[0044]

[0045] (3) reacting the compound represented by formula M2 with the compound represented by formula A to obtain methotrexate;

[0046]

[0047] Those skilled in the art will appreciate that, after step (1) and before step (2), optionally, the method further comprises step (i): dissolving the compound represented by formula M1 in water to obtain an aqueous solution of the compound represented by formula M1.

[0048] In some embodiments, step (2) is performed by:

[0049] (2-1) adjusting the pH of the aqueous solution of the compound represented by formula M1 to 5.0 to 6.0;

[0050] (2-2) adding a zinc chloride aqueous solution dropwise to the aqueous solution obtained in step (2-1) and stirring to precipitate a solid;

[0051] (2-3) Filter the mixture obtained in step (2-2) and collect the solid, which is the compound represented by formula M2.

[0052] Those skilled in the art will appreciate that in step (2-2), when the amount of precipitated solid no longer increases, it is considered the end point of step (2-2).

[0053] In some embodiments, in step (2-1), adjusting the pH of the aqueous solution of the compound represented by formula M1 to 5.0-6.0 is achieved by using hydrochloric acid.

[0054] In some embodiments, the concentration of the hydrochloric acid is 1-10mol / L (such as 1mol / L, 2mol / L, 3mol / L, 4mol / L, 5mol / L, 6mol / L, 7mol / L, 8mol / L, 9mol / L or 10mol / L, or such as 1-2mol / L, 2-3mol / L, 3-4mol / L, 4-5mol / L, 5-6mol / L, 6-7mol / L, 7-8mol / L, 8-9mol / L or 9-10mol / L), preferably 3-5mol / L.

[0055] In some embodiments, step (2-1) is performed at a temperature below 10°C.

[0056] In some embodiments, the molar concentration of the aqueous zinc chloride solution is 1-10 mol / L (such as 1 mol / L, 2 mol / L, 3 mol / L, 3.5 mol / L, 4 mol / L, 4.5 mol / L, 5 mol / L, 6 mol / L, 7 mol / L, 8 mol / L, 9 mol / L or 10 mol / L, or such as 1-2 mol / L, 2-3 mol / L, 3-3.5 mol / L, 3.5-4 mol / L, 4-4.5 mol / L, 4.5-5 mol / L, 5-6 mol / L, 6-7 mol / L, 7-8 mol / L, 8-9 mol / L or 9-10 mol / L), preferably 3-5 mol / L, such as 3.5-4 mol / L.

[0057] In some embodiments, step (2-2) is performed as follows:

[0058] (2-2-1) adding a zinc chloride aqueous solution dropwise to the aqueous solution obtained in step (2-1) to precipitate a solid;

[0059] (2-2-2) Stir the mixture obtained in step (2-2-1).

[0060] In some embodiments, step (2-2-1) is performed at a temperature below 10°C.

[0061] In some embodiments, step (2-2-2) is carried out at a temperature of 20-30°C (such as 20°C, 22°C, 24°C, 25°C, 26°C, 28°C or 30°C, or such as 20-22°C, 22-24°C, 24-25°C, 25-26°C, 26-28°C or 28-30°C).

[0062] In some embodiments, in step (2-2-2), the stirring time is 0.5-5h (such as 0.5h, 1h, 1.5h, 2h, 2.5h, 3h, 3.5h, 4h, 4.5h or 5h, or such as 0.5-1h, 1-1.5h, 1.5-2h, 2-2.5h, 2.5-3h, 3-3.5h, 3.5-4h, 4-4.5h or 4.5-5h), preferably 0.5-1.5h.

[0063] In some embodiments, steps (2-3) are performed as follows:

[0064] (2-3-1) filtering the mixture obtained in step (2-2) to obtain a filter cake;

[0065] (2-3-2) washing the filter cake with water and methanol in sequence, and collecting the filter cake to obtain a solid;

[0066] (2-3-3) Drying the solid to obtain a compound represented by formula M2.

[0067] Beneficial Effects

[0068] The invention provides a method for preparing M1. In the method, a reductive amination reaction is a one-pot reaction, which is very advantageous in terms of economy and environmental friendliness. The purity of the target product M1 prepared by the method is greater than 98.0%, the residual p-aminobenzoyl-L-glutamic acid (SM1) is less than 1.0%, which meets the quality standard (SM1<2%). The reaction system is clear, no solid is precipitated during the reaction, the product yield is high, the steps are simple, and the method is conducive to industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 : Synthetic route of methotrexate.

[0070] Figure 2-Figure 3 : The synthetic routes of methotrexate intermediates reported in existing literature.

[0071] Figure 4 : HPLC detection results of M1 (p-methylaminobenzoyl-L-glutamate sodium salt) in experimental scheme No. 16.

[0072] Figure 5 : HPLC detection results of M1 (p-methylaminobenzoyl-L-glutamate sodium salt) in experimental scheme No. 17.

[0073] Figure 6 : Example diagram of the appearance in the experimental scheme numbered 1-17.

[0074] Figure 7 :4-Aminobenzoyl-L-glutamic acid (SM1) H NMR spectrum, solvent: deuterated water.

[0075] Figure 8 : NMR hydrogen spectrum of p-aminobenzoyl-L-glutamate sodium salt (SM1 sodium salt), solvent deuterated water.

[0076] Fig. 9 :Precipitation solid NMR 1 HNMR spectrum, solvent: heavy water. DETAILED DESCRIPTION

[0077] Embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but it will be appreciated by those skilled in the art that the following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments.

[0078] In the following experimental steps, all raw materials not specifically specified can be purchased from the market. Among them, the total alkali content (purity) of sodium methoxide COA currently supplied on the market shall not be less than 98.0%, and the actual total alkali content detected is generally 96% to 98%.

[0079] The quality standard that the target product M1 needs to meet is: the starting material impurity K, i.e. SM1, needs to be controlled within 2.0%.

[0080] In order to achieve the invention purpose of this application, this application provides the following technical solutions:

[0081]

[0082] Add 25L of anhydrous methanol and 500g (1.88mol) SM1 to a clean 30L reactor, stir to dissolve, cool to 0-10°C, add 304.2g of sodium methoxide (5.64mol, 3eq) in batches, control the temperature to less than 15°C, add 253.4g of paraformaldehyde (8.44mol, 4.5eq), heat to 30-40°C and stir to react for 46-54h. Cool to 0-10°C, add 212.67g of sodium borohydride (5.62mol, 3eq) in batches, slowly heat to 40-50°C and react for 4h after the addition. After the reaction is completed, cool to 0-10°C, add 500g of water dropwise to quench the reaction, and concentrate under reduced pressure at 50-60°C until the solvent does not drip out to obtain a white solid M1 (the test results are as follows Figure 4-Figure 5 ).

[0083] The purity of the target product M1 is greater than 98.0%, the residual p-aminobenzoyl-L-glutamic acid (SM1) is less than 1.0%, which meets the quality standard, and the reaction system is clear, no solid precipitation occurs during the reaction, the product yield is high, and the steps are simple.

[0084] After the target product M1 is prepared, M2 can be further prepared by the following steps, and then used for the subsequent preparation of methotrexate.

[0085]

[0086] After obtaining the white solid M1, add 2500ml of purified water, stir until the solid is dissolved, drop to <10°C, adjust pH to 5.0-6.0 with 4N hydrochloric acid, drop zinc chloride aqueous solution (configuration: add 255g zinc chloride to 500g purified water, stir to dissolve), and a large amount of white solid is generated soon. After the dropwise addition is completed, heat to 20-30°C and stir for 1h, filter, rinse the filter cake twice with 1000g purified water, and then rinse the filter cake with 500ml methanol, collect the solid, and dry it at 60±5°C for 20h to obtain the white solid M2, which is sealed and stored.

[0087] The present invention is further explained below with reference to specific embodiments.

[0088] Example 1: Preparation of M1

[0089]

[0090] The preparation process of the compound represented by formula M1 is as follows:

[0091] Add 25L of anhydrous methanol and 500g (1.88mol) of SM1 to a clean 30L reactor, stir to dissolve, cool to 0-4°C, add sodium methoxide (X mol, Y eq) in batches, control the temperature to less than 15°C, add 253.4g (8.44mol, 4.5eq) of paraformaldehyde, heat to P°C and stir to react for Q h. Cool to 0-4°C, add 212.67g (5.62mol, 3eq) of sodium borohydride in batches, slowly heat to 45°C and react for 4h after the addition. After the reaction is completed, cool to 0-4°C, add 500g of water dropwise to quench the reaction, and concentrate under reduced pressure at 50-60°C until the solvent does not drip out to obtain a white solid M1 (sodium salt).

[0092] Among them, in the preparation process of the compound represented by the above formula M1, after heating to P℃ and stirring the reaction for Q h, the appearance of the reaction system is observed. If the reaction system is clear, no treatment is required, and the subsequent steps and reactions are directly continued. If solids are precipitated in the reaction system, the precipitated solids need to be filtered out before continuing the subsequent steps and reactions. The steps are cumbersome. Moreover, those skilled in the art can understand that when solids are precipitated, the yield of the target compound will be significantly reduced.

[0093] In addition, after the reaction was completed, HPLC was used to detect the purity of the target product M1 and the residual amount of the impurity SM1.

[0094] ⑴ Study on the temperature of imidization reaction

[0095]

[0096]

[0097] Conclusion: From the above table, it can be seen that in the imidization reaction, the reaction temperature is high, the solid is easy to precipitate, the raw material residue is large, and the yield is low. The reaction is better at a temperature of 30-40°C (such as 33°C or 38°C), the raw material residue is low, the yield is high, no solid precipitation, and the system is clear. Therefore, the subsequent imidization reaction is controlled at 30-40°C.

[0098] ⑵ Study on the dosage of sodium methoxide and the reaction time of imidization

[0099]

[0100] The appearance of the above experimental scheme is shown in the figure below: Figure 6 shown.

[0101] Conclusion: As can be seen from the above table, in the imidization reaction, the amount of sodium methoxide is a key parameter. In the imidization reaction, when the amount of sodium methoxide is greater than 3eq, solids are easily precipitated, the raw material residue is large, and the yield is low. In addition, the reaction time of the imidization reaction is also very critical. When the imidization reaction time is short (such as 19-30h), the reaction is incomplete, the raw material residue is large, and the yield is low. Therefore, in the imidization reaction, the amount of sodium methoxide is controlled to be 3eq and the reaction time is 46-54h.

[0102] In addition, the inventors have also conducted detailed studies on the solids precipitated in the experimental schemes numbered 3, 4, 5, 6, 7, 8, 9, 10, 11, 13 and 14 above, as follows:

[0103] 1) The inventors tested the precipitated solid and found that most of it was p-aminobenzoyl-L-glutamic acid sodium salt (SM1 sodium salt), see attached Figure 8-Figure 9 .

[0104] 2) After the solid was precipitated, the inventor increased the volume of the solvent methanol to 1.4 and 2 times of the original volume, and found that the solid precipitated in the early stage was basically insoluble. After that, the precipitated solid was not filtered, and the subsequent reaction was continued according to the process flow to prepare M1. The SM1 residue in the M1 finished product test seriously exceeded the standard (>>2%), and the quality was unqualified.

[0105] Example 2: Preparation of M2

[0106]

[0107] After obtaining the white solid M1 (sodium salt), add 2500ml of purified water, stir until the solid is dissolved, drop to <10°C, adjust pH to 5.0-6.0 with 4N hydrochloric acid, drop zinc chloride aqueous solution (configuration: add 255g zinc chloride to 500g purified water, stir to dissolve), and a large amount of white solid is generated soon. After the dropwise addition is completed, heat to 20-30°C and stir for 1h, filter, rinse the filter cake twice with 1000g purified water, and then rinse the filter cake with 500ml methanol, collect the solid, and dry it at 60±5°C for 20h to obtain white solid M2 (zinc salt), which is sealed and stored.

[0108] It should be understood that the invention described herein is not limited to specific methodology, experimental protocols or reagents, as these may vary. The discussion and examples provided herein are presented only to describe specific embodiments and are not intended to limit the scope of the invention, which is limited only by the claims.

Claims

1. A method for preparing a compound represented by formula M1, comprising: The compound represented by formula SM1 is subjected to a reductive amination reaction with paraformaldehyde in the presence of sodium methoxide and sodium borohydride to obtain a compound represented by formula M1 The molar ratio of the sodium methoxide to the compound represented by formula SM1 is 3:

1.

2. The method according to claim 1, wherein: The method comprises: (1-1) allowing the compound represented by formula SM1 to undergo imidization reaction with paraformaldehyde in the presence of sodium methoxide; (1-2) subjecting the imidization reaction product of step (1-1) to a reduction reaction with sodium borohydride to obtain a compound represented by formula M1; wherein, after the imidization reaction in step (1-1), the reduction reaction in step (1-2) is directly performed without any post-treatment step; Preferably, the imidization reaction of step (1-1) and the reduction reaction of step (1-2) are carried out in a one-pot process.

3. The method according to claim 2, wherein: The method further has one or more technical features selected from the following (i)-(ii): ( i) the imidization reaction of step (1-1) is carried out for 46-54 hours; (ii) The imidization reaction of step (1-1) is carried out at a temperature of 30 to 40°C.

4. The method according to any one of claims 2 to 3, wherein: The method further has one or more technical features selected from the following (i)-(vi): (i) the molar ratio of the paraformaldehyde to the compound represented by formula SM1 is 1.5:1-6:1, preferably 4.5:1; (ii) The molar ratio of the sodium borohydride to the compound represented by formula SM1 is 3:1-5:1, preferably 3:

1. (iii) the reduction reaction in step (1-2) is carried out at a temperature of 40 to 50°C, preferably 45°C; (iv) the reduction reaction of step (1-2) is carried out for 3.5 to 4.5 hours, preferably 4 hours; (v) the solvent for the imidization reaction of step (1-1) and the reduction reaction of step (1-2) is anhydrous methanol; (vi) The purity of the sodium methoxide is not less than 96%, for example, 96% to 98%.

5. The method according to any one of claims 2 to 4, wherein: Before the imidization reaction of step (1-1), the compound represented by formula SM1, the paraformaldehyde and the sodium methoxide are mixed in advance by the following method: 1) adding the compound represented by formula SM1 into a solvent and stirring to dissolve; 2) cooling the solution obtained in step 1) to 0-10° C. (preferably 0-4° C.), adding the sodium methoxide thereto, and controlling the temperature to be less than 15° C. during the addition of the sodium methoxide; 3) Adding the paraformaldehyde to the solution obtained in step 2).

6. The method according to any one of claims 2 to 5, wherein: After the imidization reaction in step (1-1) and before the reduction reaction in step (1-2), the imidization reaction product in step (1-1) and the sodium borohydride are mixed in advance by the following method: After the imidization reaction product of step (1-1) is cooled to 0-10° C. (preferably 0-4° C.), the sodium borohydride is added thereto.

7. The method according to any one of claims 2 to 6, wherein: After the reduction reaction of step (1-2), the method further comprises: (1-3) quenching the reduction reaction; (1-4) Concentrating the mixture obtained in step (1-3) under reduced pressure until no solvent is dropped out, to obtain a compound represented by formula M1; Preferably, quenching the reduction reaction is achieved by the following method: cooling the reduction reaction product of step (1-2) to 0-10° C., and adding water to quench the reduction reaction; Preferably, the reduced pressure concentration is carried out at a temperature of 50-60°C.

8. A method for preparing methotrexate, comprising: (1) Providing a compound represented by formula M1, wherein the compound represented by formula M1 is prepared by the method described in any one of claims 1 to 7; (2) acidifying and ion-exchanging the aqueous solution of the compound represented by formula M1 to obtain the compound represented by formula M2; (3) reacting the compound represented by formula M2 with the compound represented by formula A to obtain methotrexate; 9. The method according to claim 8, wherein: Step (2) is performed as follows: (2-1) adjusting the pH of the aqueous solution of the compound represented by formula M1 to 5.0 to 6.0; (2-2) adding a zinc chloride aqueous solution dropwise to the aqueous solution obtained in step (2-1) and stirring to precipitate a solid; (2-3) Filter the mixture obtained in step (2-2) and collect the solid, which is the compound represented by formula M2.

10. The method according to claim 9, wherein: The method further has one or more technical features selected from the following (i)-(v): ( i) In step (2-1), adjusting the pH of the aqueous solution of the compound represented by formula M1 to 5.0 to 6.0 is achieved by using hydrochloric acid; Preferably, the concentration of the hydrochloric acid is 1-10 mol / L; (ii) step (2-1) is carried out at a temperature below 10°C; (iii) the molar concentration of the zinc chloride aqueous solution is 1-10 mol / L; (iv) Step (2-2) is carried out as follows: (2-2-1) adding a zinc chloride aqueous solution dropwise to the aqueous solution obtained in step (2-1) to precipitate a solid; (2-2-2) stirring the mixture obtained in step (2-2-1); Preferably, step (2-2-1) is carried out at a temperature below 10°C; Preferably, step (2-2-2) is carried out at a temperature of 20 to 30°C; Preferably, in step (2-2-2), the stirring time is 0.5-5h; (v) Step (2-3) is carried out as follows: (2-3-1) filtering the mixture obtained in step (2-2) to obtain a filter cake; (2-3-2) washing the filter cake with water and methanol in sequence, and collecting the filter cake to obtain a solid; (2-3-3) Drying the solid to obtain a compound represented by formula M2.

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