A method for preparing methylene blue

Abstract

The application discloses a preparation method of methylene blue, which comprises the following steps: taking phenothiazine as a raw material, carrying out a bromination reaction to obtain 3,7-dibromo-10H phenothiazine, carrying out an acetylation reaction to obtain 3,7-dibromo-10-acetylphenothiazine, carrying out an amination reaction to obtain 3,7-bis(dimethylamino)-10-acetylphenothiazine, and finally carrying out hydrolysis and oxidation to obtain methylene blue. The application solves the technical problem of low purity of methylene blue.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and particularly relates to a method for preparing methylene blue. Background Technology

[0002] Methylene blue (Methylthioninium Chloride), chemical name: 3,7-bis(dimethylamino)phenthiazine-5-onium trihydrate, has the following structural formula:

[0003]

[0004] Methylene blue, also known as methylene blue, was first synthesized in the form of zinc oxide salt. Since 1876, methylene blue has been primarily used to treat methemoglobinemia caused by nitrites, chlorates, quinones, quinone imines, aniline, and nitrobenzene. In addition, high concentrations of methylene blue can be used to treat cyanide poisoning. In recent years, the clinical application of methylene blue has expanded to the treatment of septic and traumatic shock, cancer, and herpes zoster. Pharmacological studies have shown that methylene blue has detoxifying, analgesic, antibacterial, antiviral, and antitumor effects, and can correct hemodynamic disturbances in patients with septic shock and treat priapism.

[0005] The use of methylene blue as a medicine extends far beyond its original synthesis in 1876. With the advancement of medical research and clinical applications, its uses have been continuously expanded and optimized. From its initial treatment of methemoglobinemia to later treatment of cyanide poisoning, and then to its broader clinical applications, methylene blue plays a vital role in the medical field. With progress in scientific research, the application prospects of methylene blue may further expand, providing new possibilities for the treatment of more diseases.

[0006] Methylene blue was first synthesized by the German chemist H. Caro in 1876, and commercially produced and sold for the first time by BASF in the same year. The process involved the nitrosation of dimethylaniline, followed by reduction to N,N-dimethyl-1,4-diaminobenzene, and oxidative coupling using hydrogen sulfide and ferric chloride. Later, based on this process, several evolutionary steps were taken to produce dimethylaminoaniline from dimethylaniline via nitrosation and reduction reactions. Dimethylaminoaniline was then oxidized in sulfuric acid with thiosulfonic acid under the catalysis of zinc chloride to generate a thiosulfonic acid derivative of p-aminodimethylaniline, which subsequently underwent two oxidative cyclization reactions to produce methylene blue. The specific reaction process is as follows:

[0007]

[0008] The methylene blue produced by this process contains multiple impurities, such as Azure A and Azure B, with structures and polarities similar to methylene blue. High-purity compounds cannot be obtained through crystallization purification. Furthermore, this process uses a large amount of metal oxidants and excipients. Since methylene blue has a complexing effect on metal ions, it is difficult to obtain methylene blue that meets the pharmacopoeia's elemental impurity limits through other treatment methods. Therefore, this route is unlikely to provide high-quality pharmaceutical-grade methylene blue.

[0009] In 1997, N. Leventis et al. published an article in Tetrahedron, Volume 53, No. 29, pp. 10083-10092, on the preparation of MTC analogs from phenothiazine. Leventis et al. described a two-step process: first, phenothiazine is converted to 3,7-dibromophenothiazine-5-onium bromide in acetic acid via bromine; then, the product is reacted with a disubstituted amine in ethanol or chloroform to generate the bromide salt of the MTC analog. When using dimethylamine to prepare methylene blue, the reported yield is 70%–85%. However, this process also has some drawbacks: 1) It uses 20 molar amounts of bromine, which would result in high costs and difficulties in handling excess bromine, leading to significant environmental pressure if used for industrial production of the active pharmaceutical ingredient; 2) It uses chloroform and ethanol as solvents. Chloroform is highly toxic and unsuitable for industrial production, while ethanol readily competes with dimethylamine for nucleophilic substitution reactions, generating byproducts. This method also has certain advantages, as its raw materials, phenothiazine, dimethylamine, and bromine, are large-scale chemical products with low prices.

[0010] Building upon the work of N. Leventis et al., CN110418784A discloses a method for preparing 3,7-bis(dimethylamino)-phenothiazine-5-onium chloride or bromide. This patent uses a one-step process to convert phenothiazine into 3,7-dihalo-phenothiazine-5-onium chloride or bromide, followed by the addition of dimethylamine to convert it into the target product. This method avoids separating the intermediate brominated or chlorinated products, reducing process operations and production costs. However, due to the poor stability of 3,7-dihalo-phenothiazine-5-onium chloride or bromide, the one-step method makes it difficult to control impurities in the halogenation reaction step, potentially leading to decomposition of the halogenated product. This results in a product with poor quality, high impurity levels, and fails to meet pharmaceutical requirements.

[0011] CMWischik et al. disclosed a method for purifying methylene blue, capable of purifying commercially available methylene blue with a purity of less than 95% to a high purity level. First, methylene blue is reduced to leucomycin with sodium borohydride (or alternatively hydrazine hydrate and methylhydrazine). Then, acylation is performed using acetic anhydride. The acylated product is purified by crystallization, followed by hydrolysis and oxidation to obtain high-purity methylene blue. We conducted reproducibility experiments on this preparation method and found that the yields of 3,7-bis(dimethylamino)-10-acetyl-phenothiazine prepared by the reaction and the yield of methylene blue prepared by hydrolysis and oxidation were both around 50%, close to the examples in the patent. The overall yield of the purification steps was approximately 25%, which is low. Furthermore, leucomycin, due to its strong reducing properties, is easily oxidized to methylene blue by air, making it unsuitable for extraction as a standalone intermediate. The main advantage of this method is that the 3,7-bis(dimethylamino)-10-acetyl-phenthiazide prepared loses the complexing effect of methylene blue on metal ions, which has a positive effect on removing elemental impurities. In addition, the polarity of the acetylation product is reduced, making it easier to crystallize and purify to obtain high-purity methylene blue. The main disadvantage is the low yield, with the molar yield of the acetylation step being only about 50%.

[0012]

[0013] Currently, methylene blue is used in tablet or injection form. To adapt to a wider range of applications, it is necessary to provide raw materials with high chemical purity and low heavy metal content to meet the efficacy and safety requirements of the drug as much as possible. Summary of the Invention

[0014] This invention provides a method for preparing methylene blue, thereby solving the technical problem of low purity of methylene blue.

[0015] In view of this, the present invention provides a method for preparing methylene blue, comprising the following steps: using phenothiazine as a raw material, brominating it to obtain 3,7-dibromo-10H-phenothiazine, then acetylifying it to obtain 3,7-dibromo-10-acetylphenothiazine, then ammonifying it to obtain 3,7-bis(dimethylamino)-10-acetylphenothiazine, and finally hydrolyzing and oxidizing it to obtain methylene blue.

[0016] Furthermore, the process route for methylene blue is as follows:

[0017]

[0018] Optionally, the methylene blue is prepared by the following method: 3,7-bis(dimethylamino)-10-acetylphenthiazide is added to an aqueous hydrochloric acid solution, heated to react, and after the reaction is complete, the temperature is lowered, and then an aqueous ferric chloride solution is added to continue the reaction. Then the mixture is filtered, washed, and dried to obtain methylene blue.

[0019] Furthermore, the methylene blue is prepared by the following method: 3,7-bis(dimethylamino)-10-acetylphenthiazide and water are added to a 2000 mL reaction flask, followed by concentrated hydrochloric acid. The mixture is heated to 70-100 °C and reacted for 0.8-1.2 h. The temperature is then lowered to 0-30 °C, and 1.2 or 2.0 eq of ferric chloride aqueous solution is added. The reaction continues for another 0.8-1.2 h. The mixture is then filtered, washed with a 1% hydrochloric acid aqueous solution, washed with water, and dried to obtain methylene blue.

[0020] The amount of water added to each 6g of 3,7-bis(dimethylamino)-10-acetylphenothiazine is 10-80mL, the amount of hydrochloric acid added to each 1g of 3,7-bis(dimethylamino)-10-acetylphenothiazine is 1.5-3mL, 1.2eq of ferric chloride aqueous solution is made by dissolving 59.2g of ferric chloride hexahydrate in 177.6g of water, and 2.0eq is made by dissolving 98.7g of ferric chloride hexahydrate in 296g of water.

[0021] Optionally, the 3,7-dibromo-10H-phenothiazine is prepared by the following method: phenothiazine is added to acetonitrile, bromine is added at a certain temperature, after the addition is complete, the reaction is carried out, sodium bisulfite solution or sodium sulfite solution is added to the reaction system, filtered, and washed to obtain 3,7-dibromo-10H-phenothiazine.

[0022] Furthermore, the 3,7-dibromo-10H-phenthiazide is prepared by the following method: phenthiazide and acetonitrile are added to a 2000 mL reaction flask, bromine is added at a certain temperature, and after the addition is complete, the reaction is allowed to proceed for 1-2 hours. After the reaction is completed, sodium bisulfite solution or sodium sulfite solution is added to the reaction system, filtered, and washed with water to obtain 3,7-dibromo-10H-phenthiazide.

[0023] The acetonitrile added per 1g of phenothiazine is 10-20mL, and the sodium bisulfite added per 1g of phenothiazine is 2-4g or 2.5-4.5g of sodium bisulfite, which are then prepared into an aqueous solution.

[0024] Optionally, the weight ratio of the phenothiazine to bromine is 1:(1.6-3.2).

[0025] Optionally, the temperature is 20-30°C.

[0026] Optionally, the 3,7-dibromo-10-acetylphenothiazine is prepared by the following method: 3,7-dibromo-10H-phenothiazine is added to a first solvent, followed by an acylation reagent, and the reaction is carried out. After the reaction is complete, water is added, and the mixture is filtered, washed, and dried to obtain 3,7-dibromo-10-acetylphenothiazine.

[0027] Optionally, the first solvent is either DMF or acetonitrile, and the acylation reagent is either acetyl chloride or acetic anhydride.

[0028] Furthermore, the 3,7-dibromo-10-acetylphenthiazide is prepared by the following method: 3,7-dibromo-10H-phenthiazide and DMF are added to a 2000 mL reaction flask, followed by the addition of triethylamine. Acetyl chloride is then added at a temperature of -10 to 0 °C. After completion, the reaction is maintained at the current temperature for 1.5-2.5 h. Water is added, the mixture is filtered, washed with water, and dried to obtain 3,7-dibromo-10-acetylphenthiazide; wherein, The amount of DMF added per 1g of 3,7-dibromo-10H-phenothiazine is 4-6mL, the weight ratio of 3,7-dibromo-10H-phenothiazine to triethylamine is 1:(0.3387-0.3388), the weight ratio of 3,7-dibromo-10H-phenothiazine to acetyl chloride is 1:(0.23-0.25), and the amount of water added per 1g of 3,7-dibromo-10H-phenothiazine is 18-22mL.

[0029] Furthermore, the 3,7-dibromo-10-acetylphenothiazine is prepared by the following method: 3,7-dibromo-10H-phenothiazine and acetonitrile are added to a 2000 mL reaction flask, followed by the addition of acetic anhydride. After completion, the mixture is heated to 90-110 °C and reacted for 4-8 h. Water is added, the mixture is filtered, washed with water, and dried to obtain 3,7-dibromo-10-acetylphenothiazine. The amount of acetonitrile added per 1 g of 3,7-dibromo-10H-phenothiazine is 3.7-3.8 mL, the weight ratio of 3,7-dibromo-10H-phenothiazine to acetic anhydride is 1:(1.42-1.43), and the amount of water added per 1 g of 3,7-dibromo-10H-phenothiazine is 10-15 mL.

[0030] Optionally, the 3,7-bis(dimethylamino)-10-acetylphenothiazine is prepared by the following method: 3,7-dibromo-10-acetylphenothiazine is added to a second solvent, followed by an aqueous solution of dimethylamine and cuprous iodide. The mixture is heated to allow the reaction to proceed. After the reaction is complete, the mixture is cooled, water is added, crystals are precipitated, and the crystals are filtered, washed, and dried.

[0031] 3,7-Di(dimethylamino)-10-acetylphenthiazide was obtained.

[0032] Furthermore, the 3,7-bis(dimethylamino)-10-acetylphenothiazine is prepared by the following method: 3,7-dibromo-10-acetylphenothiazine and a second solvent are added to a 2000 mL reactor, followed by dimethylamine aqueous solution and cuprous iodide. The mixture is heated to 100-150 °C and reacted for 10-20 h. After the reaction is complete, the temperature is lowered to 5-50 °C, water is added, and crystallization is allowed to occur for 0.8-1.2 h. The mixture is then filtered, washed with water, and dried to obtain 3,7-bis(dimethylamino)-10-acetylphenothiazine.

[0033] The amount of the second solvent added to each 1g of 3,7-dibromo-10-acetylphenothiazine is 5-6.25mL, the mass fraction of the dimethylamine aqueous solution is 40%, the weight ratio of 3,7-dibromo-10-acetylphenothiazine and cuprous iodide is 1:(0.0075-0.01), and the amount of water added to each 1g of 3,7-dibromo-10-acetylphenothiazine is 10-15mL.

[0034] Optionally, the weight ratio of the aqueous solution of 3,7-bis(dimethylamino)-10-acetylphenthiazide and dimethylamine is 1:(1.14-2.28).

[0035] Optionally, the second solvent is either NMP or DMF.

[0036] As can be seen from the above technical solutions, the embodiments of the present invention have the following advantages:

[0037] 1. The total yield of methylene blue of pharmaceutical grade using phenothiazine as raw material in this process can reach over 94%, the purity can reach over 99.6%, and the content of azurite B can be reduced to below 0.3%. In terms of overall quality and yield, it is far superior to the existing process level.

[0038] 2. The use of bromine and dimethylamine has been reduced, which not only lowers production costs but also reduces environmental pressure. It is environmentally friendly because it does not use highly toxic solvents such as chloroform.

[0039] 3. Since methylene blue has the effect of complexing metal ions, if a large amount of materials containing metal ions are used in the process, the elemental impurities in the finished product may exceed the pharmacopoeia limits (the European Pharmacopoeia has clear regulations). However, in the steps of this application, only two materials containing metal elements, ferric chloride and cuprous iodide, were used. The test results showed that the metal elements in this product meet the pharmacopoeia quality standards. Therefore, it is shown that the methylene blue prepared by this process meets the pharmaceutical use requirements. Attached Figure Description

[0040] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0041] Figure 1 This is the hydrogen spectrum of the prepared methylene blue.

[0042] Figure 2 This is the liquid phase diagram of the prepared methylene blue. Detailed Implementation

[0043] To enable those skilled in the art to better understand the present invention, the technical solutions in the embodiments of the present invention are clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, all raw materials, reagents, instruments, and equipment used in the present invention can be purchased on the market or prepared by existing methods.

[0044] Preparation Example

[0045] Preparation Example 1

[0046] A 3,7-dibromo-10H-phenthiazide is prepared by the following method:

[0047] 50 g of phenothiazine and 500 mL of acetonitrile were added to a 2000 mL reaction flask. 80 g of bromine was added at 25 °C. After the addition was complete, the reaction was allowed to proceed for 1 h. After the reaction was completed, 208 g of an aqueous solution of sodium bisulfite was added to the reaction system. The mixture was filtered and washed with water to obtain 3,7-dibromo-10H-phenothiazine. 86.5 g was recovered, with a yield of 96%.

[0048] Preparation Example 2

[0049] A 3,7-dibromo-10H-phenthiazide is prepared by the following method:

[0050] 50 g of phenothiazine and 1000 mL of acetonitrile were added to a 2000 mL reaction flask. 160 g of bromine was added at 25 °C. After the addition was complete, the reaction was allowed to proceed for 2 h. After the reaction was completed, 500 g of an aqueous solution of sodium sulfite was added to the reaction system. The mixture was filtered and washed with water to obtain 3,7-dibromo-10H-phenothiazine. 87.1 g was obtained, with a yield of 96.7%.

[0051] Preparation Example 3

[0052] A 3,7-dibromo-10-acetylphenthiazide is prepared by the following method:

[0053] 80 g of 3,7-dibromo-10H-phenthiazide prepared in Preparation Example 2 and 400 mL of LDM were added to a 2000 mL reaction flask, followed by 27.1 g of triethylamine. 19.2 g of acetyl chloride was then added at -5 °C. After the reaction was complete, the mixture was kept at the current temperature for 2 h. 1600 mL of water was added, the mixture was filtered, washed with water, and dried to obtain 83.1 g of 3,7-dibromo-10-acetylphenthiazide, with a yield of 93.0%.

[0054] Preparation Example 4

[0055] A 3,7-dibromo-10-acetylphenthiazide is prepared by the following method:

[0056] 80 g of 3,7-dibromo-10H-phenthiazide prepared in Preparation Example 2 and 300 mL of acetonitrile were added to a 2000 mL reaction flask, followed by 114 g of acetic anhydride. After the reaction was completed, the mixture was heated to 100 °C and reacted for 6 h. 1000 mL of water was added, the mixture was filtered, washed with water, and dried to obtain 80.7 g of 3,7-dibromo-10-acetylphenthiazide, with a yield of 90.3%.

[0057] Preparation Example 5

[0058] A 3,7-bis(dimethylamino)-10-acetylphenthiazide is prepared by the following method:

[0059] 80 g of 3,7-dibromo-10-acetylphenothiazine prepared in Preparation Example 3 and 400 mL of NMP were added to a 2000 mL reactor, followed by 91.2 g of 40% dimethylamine aqueous solution and 0.8 g of cuprous iodide. The mixture was heated to 135 °C and reacted for 20 h. After the reaction was completed, the temperature was lowered to 30 °C, 1000 mL of water was added, and crystallization was allowed to occur for 1 h. The mixture was then filtered, washed with water, and dried to obtain 62.5 g of 3,7-di(dimethylamino)-10-acetylphenothiazine, with a yield of 95.1%.

[0060] Preparation Example 6

[0061] A 3,7-bis(dimethylamino)-10-acetylphenthiazide is prepared by the following method:

[0062] 80 g of 3,7-dibromo-10-acetylphenothiazine prepared in Preparation Example 3 and 500 mL of LDM were added to a 2000 mL reactor, followed by 182.4 g of a 40% dimethylamine aqueous solution and 0.6 g of cuprous iodide. The mixture was heated to 135 °C and reacted for 10 h. After the reaction was completed, the temperature was lowered to 30 °C, 1000 mL of water was added, and crystallization was allowed to occur for 1 h. The mixture was then filtered, washed with water, and dried to obtain 61.6 g of 3,7-di(dimethylamino)-10-acetylphenothiazine, with a yield of 93.7%.

[0063] Example

[0064] Example 1

[0065] A method for preparing methylene blue includes the following steps:

[0066] 60 g of 3,7-bis(dimethylamino)-10-acetylphenthiazide prepared in Preparation Example 5 and 600 mL of water were added to a 2000 mL reaction flask, followed by 180 mL of concentrated hydrochloric acid. The mixture was heated to 90 °C and reacted for 1 h. The temperature was then lowered to 25 °C, and 2 eq of ferric chloride solution (98.7 g of ferric chloride hexahydrate dissolved in 296 g of water) was added. The reaction was continued for 1 h. The mixture was filtered, washed with a 1% hydrochloric acid aqueous solution, washed with water, and dried to obtain 67.4 g of methylene blue, with a yield of 99%, a purity of 99.67%, and an azure B content of 0.28%.

[0067] Example 2

[0068] A method for preparing methylene blue includes the following steps:

[0069] 60 g of 3,7-bis(dimethylamino)-10-acetylphenthiazide prepared in Preparation Example 5 and 800 mL of water were added to a 2000 mL reaction flask, followed by 90 mL of concentrated hydrochloric acid. The mixture was heated to 75 °C and reacted for 1 h. The temperature was then lowered to 5 °C, and 1.2 eq of ferric chloride solution (59.2 g of ferric chloride hexahydrate dissolved in 177.6 g of water) was added. The reaction was continued for 1 h. The mixture was filtered, washed with a 1% hydrochloric acid aqueous solution, washed with water, and dried to obtain 64.5 g of methylene blue, with a yield of 94.7%, a purity of 99.71%, and an azure B content of 0.25%.

[0070] Performance testing

[0071] The finished methylene blue from Example 1 was subjected to the following performance tests and compared with the EP 11.0 limit. The test results are shown in Table 1.

[0072] Table 1 Test Results

[0073]

[0074]

[0075] As can be seen from Examples 1-2 and Table 1, the methylene blue prepared by the method of this application reduces the amount of bromine and dimethylamine used, which not only reduces production costs but also reduces environmental pressure. It does not use highly toxic solvents such as chloroform, making it environmentally friendly. Furthermore, the methylene blue prepared by this application has superior quality, higher purity, and higher yield, with a yield of over 94%, a purity of over 99.6%, and azurite B content reduced to below 0.3%. Considering both quality and yield, it is far superior to the existing process level.

[0076] The above-described embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A process for the preparation of methylene blue, characterized in that: The method comprises the following steps: adding phenothiazine into acetonitrile, adding bromine at a temperature of 20-30°C, adding sodium bisulfite solution or sodium sulfite solution into the reaction system after the reaction is completed, filtering, washing, and obtaining 3,7-dibromo-10H phenothiazine; adding 3,7-dibromo-10H phenothiazine into a first solvent, adding acylating agent, reacting, adding water after the reaction is completed, filtering, washing, and drying to obtain 3,7-dibromo-10-acetyl phenothiazine; adding 3,7-dibromo-10-acetyl phenothiazine into a second solvent, adding dimethylamine aqueous solution and cuprous iodide, heating, reacting, cooling after the reaction is completed, adding water, crystallizing, filtering, washing, and drying to obtain 3,7-bis(dimethylamino)-10-acetyl phenothiazine; adding 3,7-bis(dimethylamino)-10-acetyl phenothiazine into hydrochloric acid aqueous solution, heating, reacting, cooling after the reaction is completed, adding ferric trichloride aqueous solution, continuously reacting, filtering, washing, and drying to obtain methylene blue.

2. The method of claim 1, wherein: The weight ratio of the phenothiazine and bromine is 1: (1.6-3.2).

3. The method for preparing methylene blue according to claim 1, characterized in that: The first solvent is any one of DMF and acetonitrile, and the acylating agent is any one of acetyl chloride and acetic anhydride.

4. The method for preparing methylene blue according to claim 1, characterized in that: The weight ratio of the 3,7-bis(dimethylamino)-10-acetyl phenothiazine and dimethylamine aqueous solution is 1: (1.14-2.28).

5. The method for preparing methylene blue according to claim 1, characterized in that: The second solvent is any one of NMP and DMF.

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