Process for the Purification of Methylene Blue

Abstract

The present application provides method for producing methylene blue that includes the steps of providing a reaction mixture having one or more methylene blue intermediates; precipitating metal from the reaction mixture; and producing therefrom crude methylene blue. The crude may further be purified, resulting in methylene compounds having low impurities, preferably having a purity greater than about 97%, having Azure B impurity no greater than 2.5%, and/or a total metal content no greater than 77 ppm. Formulations containing such compounds are also provided.

Claims

1. A compound comprising methylene blue having a purity greater than about 97%.

2. The compound of claim 1, the methylene blue further having Azure B impurity no greater than 2.5%.

3. The compound of claim 1, the methylene blue further having total metal content no greater than 77 ppm.

4. A method for producing methylene blue comprising: providing a reaction mixture comprising one or more methylene blue intermediates; precipitating metal from the reaction mixture; and producing therefrom crude methylene blue.

5. The method of claim 3, comprising extracting a solvent from the reaction mixture, wherein the step of precipitating the metal from the reaction mixture is performed during solvent extraction.

6. The method of claim 5, wherein solvent extraction is performed with the reaction mixture having a pH between about pH 9 and about pH 11.

7. The method of claim 3, wherein methylene blue crude is produced in-situ without isolation of any intermediate(s).

8. The method of claim 3, wherein providing the reaction mixture comprises: reacting N,N-dimethylaniline with sodium nitrite in hydrochloric acid to obtain an in-situ nitroso compound; reducing the in-situ nitroso compound in the presence of zinc and hydrochloric acid to get obtain in-situ p-amino dimethyl aniline; reacting the in-situ p-amino dimethyl aniline with sulfuric acid and zinc chloride in a presence of sodium thiosulfate pentahydrate, sodium dichromate, and aluminium sulphate octahydrate to obtain an in-situ thiosulfonic acid compound; treating the in-situ thiosulfonic acid compound with another molecule of N,N-dimethylaniline in a presence of sodium dichromate and zinc chloride to yield in-situ Di-cyclic compound; and allowing the in-situ Di-cyclic compound to undergoes a cyclisation reaction in presence of manganese oxide to yield methylene blue crude.

9. The method of claim 3, comprising purifying the methylene blue crude to obtain methylene blue having a purity greater than about 97%.

10. The method of claim 3, comprising purifying the methylene blue crude to obtain methylene blue having Azure B impurity no greater than 2.5%.

11. The method of claim 3, comprising purifying the methylene blue crude to obtain methylene blue having total metal content no greater than 77 ppm.

12. The method of claim 3, comprising purifying the methylene blue crude in water and tetrahydrofuran, followed by treatment with hydrochloric acid to get stable methylene blue having a pH in range of 3.0-4.5.

13. The method of claim 3, comprising adjusting the reaction mixture to a pH of less than about 2.

14. The method of claim 3, comprising purifying the methylene blue crude using a polar solvent to obtain methylene blue.

15. The method of claim 14, wherein the polar solvent comprises acetic acid.

16. A formulation comprising methylene blue prepared according to the method of claim 1.

17. The formulation of claim 16, wherein the methylene blue has a purity greater than about 97%.

18. The formulation of claim 16, wherein the methylene blue has Azure B impurity no greater than 2.5%.

19. The formulation of claim 16, wherein the methylene blue has a total metal content no greater than 77 ppm.

20. The formulation of claim 16, having a concentration of methylene blue from about 1 mg/ml to about 20 mg/ml.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0048] FIG. 1 illustrates a characteristic X-ray powder diffraction pattern of methylene blue form A obtained using the processes disclosed herein.

[0049] FIG. 2 illustrates a characteristic DSC pattern of methylene blue form A obtained using the processes disclosed herein.

[0050] FIG. 3 illustrates a characteristic TGA pattern of methylene blue form A obtained using the processes disclosed herein.

[0051] FIGS. 4A-4B illustrate processes for the preparation of methylene blue according to at least one embodiment of the processes disclosed herein.

[0052] For the purposes of illustration, drawings represent preferred embodiments, it being understood, however, that the invention is not limited to the precise embodiments shown therein.

DETAILED DESCRIPTION

[0053] Important aspects in the preparation of Methylene blue are quality and production costs of the end product. Owing to regulatory requirements, high quality standards have to be met. Of interest in this context are purity and content of the active compound. Coupled to purity, it is in particular the spectrum of by-products which need to be monitored. Minor components have to be toxicologically qualified and assessed. Accordingly, they are listed in specifications and the maximum occurrence in the product is defined. For reasons of product safety and for the good of the patient, the by-product spectrum and the presence of individual contaminants are kept as low as possible to achieve the desired result.

[0054] Referring to FIG. 4A, in a first embodiment, the present application provides an improved process for the preparation of crude methylene blue. The process for the preparation of methylene blue crude according to this embodiment is performed as in-situ without isolation of any intermediate(s). The process for the preparation methylene blue intermediate generally includes the reaction of:

[0055] a. N,N-dimethylaniline with sodium nitrite in hydrochloric acid to obtain in-situ nitroso compound.

[0056] b. the in-situ nitroso compound is further reduced in the presence of zinc and hydrochloric acid to obtain in-situ p-amino dimethyl aniline.

[0057] c. the in-situ p-amino dimethyl aniline is further reacted with sulfuric acid and zinc chloride in presence of sodium thiosulfate pentahydrate, sodium dichromate, and aluminium sulphate octahydrate to obtain in-situ thiosulfonic acid compound.

[0058] d. the in-situ thiosulfonic acid compound is further treated with another molecule of N,N-dimethylaniline in presence of sodium dichromate and zinc chloride to yield in-situ Di-cyclic compound.

[0059] e. the in-situ Di-cyclic compound then undergoes a cyclisation reaction in presence of manganese oxide to yield methylene blue crude.

[0060] Referring to FIG. 4B, in another embodiment the process further involves the purification of methylene blue crude to get methylene blue, preferably having less metal content as compared to known processes. In one embodiment, the process involves a purification of methylene blue crude in water and tetrahydrofuran followed by treatment with hydrochloric acid to get a stable methylene blue having a pH in range of 3.0-4.5, which remains stable in stability study.

[0061] Methylene blue obtained by this process is in crystalline form A. Characterisation details with XRD (x-ray diffraction), DSC (differential scanning calorimetry) and TGA (thermal gravimetric analysis) data for form A is depicted in the drawings (FIGS. 1-3). DSC of crystalline form A is 191.24° C. Methylene blue obtained by this process is pentahydrate.

[0062] Four major impurities have been known which are affecting the quality and yield of the methylene blue in the synthesis. These include 1. Azure A, 2. Azure B, 3. Azure C, and 4. Methylene Violet Bernthsen (MVB). The structures for individual impurity are depicted in Table 1.

TABLE-US-00001 TABLE 1 [00003] [00004] [00005] [00006]

[0063] Formation of an above impurity is observed in the basic condition. Use of inorganic salts in the process for producing methylene blue are the source for the relatively high metal content. Thus, there is an urgent need to develop a process which controls or otherwise limits the metal content in the product, which ultimately improves the purity with minimum genotoxic impurity.

[0064] Metal content can be removed by precipitating the metal from the reaction mixture. Prior processes proposed the extraction thereof in acidic condition, which does not give good results. The present application provides a process for producing methylene blue which reduces the metal content therein by precipitation during solvent extraction process in the basic condition.

[0065] Solvent extraction is done in the basic condition preferably between about pH 9 to about pH 11, more preferably above about pH 10. In a preferred embodiment, extraction is performed by using methylene dichloride at about pH 10.0 to about 10.5. Suitable base(s) required for the adjustment of pH to above about 9 includes but is not limited to sodium carbonate, sodium bicarbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, ammonia, and triethylamine.

[0066] Another area of concern is pH of formulated methylene blue. USP mentions that pH of methylene blue formulation should be in between about 3.0 to about 4.5. In formulation, pH of Methylene blue solution tends to increase drastically when the pH of the active pharmaceutical ingredient (API) is kept in between about 3.0 to about 4.5 as per prior art process. To overcome such a problem, the reaction mass pH is preferably kept less than about 2 and more preferably pH of less than about 1. Suitable acids for the adjustment of pH to below about 1 includes, but is not limited to, hydrochloric acid.

[0067] Following the process discussed in Fundamental Processes of Dye Chemistry (above) to synthesize methylene blue, one unknown impurity is observed at 0.94 RRT (close to methylene blue peak), as per the USP method, with a range of 0.5% to 1%. The impurity has been isolated and well analysed with mass of 316.1 g/mol. A crystallisation process is provided to reduce the impurity content below about 0.1%. The crude may be purified in polar solvent to remove the impurity at 0.94 RRT. The polar solvent used may be selected from Acetic acid and in the presence of conc HCl. Anti-solvent may be used for the isolation of product. Anti-solvents used may be selected from acetone, acetonitrile, THF, DMF, IPA, DIPE or DMSO, or a combination thereof. In a preferred embodiment, acetic acid is used for removal of impurity at 0.94 RRT.

[0068] The isolation of crude intermediate may be induced using techniques which include but are not limited to concentrating, cooling, separation, stirring, shaking, combining with an anti-solvent, adding seed crystals, evaporation, flash evaporation, simple evaporation, rotational drying, or the like.

[0069] Once the methylene blue is obtained in isolated, drying can be carried out in a tray dryer, vacuum oven, air oven, cone vacuum dryer, rotary vacuum dryer, fluidized bed dryer, spin flash dryer, flash dryer, or the like. The drying can be carried out at temperatures of less than about, less than about 40° C., less than about 30° C., or any other suitable temperatures; at atmospheric pressure or under a reduced pressure; as long as the crystalline pharmaceutical ingredient is not degraded in its quality. The drying can be carried out for any desired times until the required product quality is achieved. Suitable time for drying can vary from few minutes to several hours for example from about 30 minutes to about 10 or more hours.

[0070] Formulation containing methylene blue may then be prepared by using this isolated material. Process for the preparation for methylene blue composition can be selected from any suitable process. Formulation can be either in solid dosage form or liquid dosage form. In a preferred embodiment, liquid dosage form is prepared. The formulation may have a concentration from about 1 mg/ml to about 20 mg/ml or any suitable concentration.

[0071] Methylene blue product obtained by this process is then loaded for stability at about 20-25° C. and at about 40° C. Purity and metal content of Methylene blue is tested for up to six months.

[0072] Stability data for Methylene blue is depicted in Table 2 & 3

TABLE-US-00002 TABLE 2 Stability data at 25° C. Trial No. Trial 1 Trial 2 Trial 3 Profile 6M 25/60 6M 25/60 6M 25/60 Purity 97.54 97.6 97.56 Azure A 0.03 0.08 0.04 Azure B 2.37 2.2 2.17 Azure C ND 0.01 ND Highest Unknown 0.04 0.06 0.08 Total impurities 2.46 2.4 2.44 Metal Content (ppm) Al 49.1 47.57 8.31 Cr 11.68 1.79 1.76 Cu 1.91 0.74 0.7 Mn 0.68 0.61 0.48 Zn 13.14 24.89 46.16 Total metal 76.51 75.6 57.41 content(ppm)

TABLE-US-00003 TABLE 3 Stability data at 40° C. Trial No. Trial 1 Trial 2 Trial 3 Profile 6M 40/75 6M 40/75 6M 40/75 Purity 97.5 97.54 97.48 Azure A 0.03 0.08 0.04 Azure B 2.41 2.26 2.26 Azure C ND 0.01 ND Highest Unknown 0.03 0.06 0.07 Total impurities 2.5 2.46 2.52 Metal Content (ppm) Al 46.2 38.54 13.71 Cr 12.57 3.26 1.54 Cu 1.13 0.07 0.83 Mn 0.49 0.49 0.51 Zn 14.58 20.55 45.89 Total metal 74.97 62.91 62.48 content(ppm)

[0073] The Methylene blue intermediate synthesized by this route has advantageous properties selected from at least one of: chemical purity, stability—such as storage stability, stability to pH, stability to dehydration, stability to polymorphic conversion, flowability, solubility, morphology or crystal habit, low hygroscopicity and low content of residual solvents. In a preferred embodiment, the methylene blue produced has a purity greater than about 97% and Azure B impurity no greater than between 2.0-2.5%.

[0074] Certain specific aspects and embodiments of the present application will be explained in more detail with reference to the following examples, which are provided only for purposes of illustration and should not be construed as limiting the scope of the present application in any manner.

EXPERIMENTAL SECTION

[0075] Preparation of Methylene Blue Crude

[0076] Step 1a:

[0077] In a round bottom flask (RBF) water (1.86 lit) was added. The solution was cooled to approximately 5° C. N,N-dimethylaniline (300 g) and HCl (37%)(0.9 lit) was charged into the cooled water. To this mixture was added dropwise an aqueous solution of sodium nitrite (0.18 kg) in water (0.9 lit). The resulting suspension was stirred at a low temperature (0-5° C.) for 4 hours.

[0078] Step 1b:

[0079] Charged hydrochloric acid solution into the above reaction mass at 0 to 5° C. Zinc dust powder (0.70 kg) was added in one aliquot portions. The mixture was stirred for 2 hours at 0 to 5° C. The mixture was filtered through celite bed.

[0080] Step 1c:

[0081] The filtrate ml was collected and cooled to 0 to 5° C. in RBF. Sulfuric acid (27 ml) was added into the reaction mass at 0 to 5° C. Charged zinc chloride solution (30 gm) in (0.3 lit) water at 0 to 5° C. Charged aluminum sulphate solution (0.25 kg) in (0.75 lit) water at 0 to 5° C. Charged sodium thiosulphate solution (0.72 kg) in water (0.6 lit) at 0 to 5° C. Added sodium dichromate solution (0.234 kg) in process water (0.36 lit) into the reaction mass under vigorous stirring at 0 to 5° C. The solution was then stirred at low temperature (at 0 to 5° C.) for 2 hours.

[0082] Step 1d:

[0083] A homogenous solution of N,N-dimethylaniline (0.25 lit), water (0.36 lit) and HCl (0.33 lit) was then added to the chilled reaction mass. Then a solution of sodium dichromate dihydrate (0.45 kg) in water (0.75 lit) was added dropwise at 0 to 5° C. Raise the reaction mass temperature to 25 to 30° C. and stir the reaction mass for 1.0 hr. at 25 to 30° C. Heated the reaction mass up to 70° C. to 75° C.

[0084] Step 1e:

[0085] Charged manganese dioxide solution (0.15 kg) in process water (0.36 lit) into the reaction mass under vigorous stirring at 70° C. to 75° C. Further heat the reaction mass up to 80° C. to 85° C. Stirred the reaction mass for 6.0 hrs at 80° C. to 85° C. A deep blue colour was formed. Cooled the reaction mass to 25° C. to 30° C. Filtered the reaction mass though Büchner funnel and discarded filtrate ml.

[0086] Wet cake was taken in RBF and methanol (2×2.4 lit) was added. Reaction mass was filtered. Charged wet cake in RBF and make slurry with water (2×5.1 lit). The filtrate was collected and treated with sodium chloride (1.0 kg). The mixture was stirred until the deep blue colour disappeared. The mixture was then filtered and wash with tetrahydrofuran (300 ml).

[0087] Wet cake was charged and dissolved in (10.5 lit) process water and adjusted the reaction mass pH 9.0 to 10.0 with sodium carbonate solution (15%) at 20° C. to 30° C. Stirred the reaction mass for 15 minutes at 25° C. to 30° C. Filtered the reaction mass though celite bed, charged filtrate ml and slowly adjusted the reaction mass pH 3.0 to 7.0 with dil. hydrochloric acid solution at 20° C. to 30° C. Charged sodium chloride (1.0 kg) into the reaction mass under stirring and stir for 10 minutes at 25° C. to 30° C. Filtered and washed with tetrahydrofuran (2×250 ml). Charged process water (18.0 lit) and wet cake into RBF at 25° C. to 30° C. Adjusted the reaction mass pH between 10.00 to 10.50 by using sodium carbonate solution (15%) and washed the aqueous layer with methylene dichloride (5.1 lit) for five times. Took aqueous layer and adjusted pH 2.0 to 4.0 with diluted hydrochloric acid solution at 25° C. to 30° C. and treated with sodium chloride (1.0 kg). The mixture was stirred until the deep blue colour disappeared. The mixture was filtered and wash with tetrahydrofuran (300 ml).

[0088] Charged acetic acid (900 ml), wet cake and conc. hydrochloric acid (1050 ml). Slowly charged acetone (3 lit) into the reaction mass. Stirred the reaction mass for 2.0 hrs. at 25° C. to 35° C. Filtered and washed with tetrahydrofuran (2×150 ml). Charged process water (18.0 lit) and wet cake into RBF and stir for 15 minutes at 25 to 30° C. Charged sodium chloride (1.0 kg) in reaction and mixture was stirred until the deep blue colour disappeared. The mixture was filtered and washed with tetrahydrofuran (300 ml).

[0089] Charged process water (18.0 lit) and wet cake into RBF at 25° C. to 30° C. Adjust the reaction mass pH between 10.00 to 10.50 by using sodium carbonate solution (15%) and wash the aqueous layer with methylene dichloride (5.1 lit) for five times. Took aqueous layer and adjust pH 2.0 to 4.0 with dilute hydrochloric acid solution at 25° C. to 30° C. and treated with sodium chloride (1.0 kg). The mixture was stirred until the deep blue colour disappeared. The mixture was filtered and wash with tetrahydrofuran (300 ml). Charged wet cake and process water (15 lit) and adjust pH 1.5 to 2.0 with dilute hydrochloric acid solution at 25° C. to 30° C. and treated with sodium chloride (1.0 kg). The mixture was stirred until the deep blue colour disappeared. The mixture was filtered and washed with tetrahydrofuran (2×250 ml). Dried the material in vacuum at 40° C. to 45° C. to obtain Dry wt.=153 gm and Yield: 37.87%.

[0090] Preparation of Methylene Blue:

[0091] Step 2:

[0092] Stage-I (methylene blue crude, 150 gm) was taken in methanol (1.5 Lit) and reaction mass was stirred at 25° C. to 35° C. to get homogeneous solution. Reaction mass was filtered through 0.45 μl and then filtrate mL cooled to get 0° C. to 5° C. Conc. hydrochloric acid (150 mL) was slowly added into the reaction mass at 0° C. to 5° C. Reaction mass was stirred for 10 to 20 minutes at 0° C. to 5° C. Fine filtered Tetrahydrofuran (1.5 lit) was added into reaction mass and stirred for 30 minutes at 0° C. to 5° C. Reaction mass was filtered and washed with fine filtered Tetrahydrofuran (300 mL). Wet cake was taken in fine filtered process water (750 ml) and stirred for 30 minutes at 25° C. to 30° C. Fine filtered tetrahydrofuran (1.5 lit) was charged into reaction mass at 25° C. to 30° C. Reaction mass stirred for 1.0 hr. at 25° C. to 30° C. Reaction mass was filtered and washed with fine filtered Tetrahydrofuran (2×150 mL). Material was dried in vacuum oven at 30° C. to 35° C. for 2.0 hrs.

[0093] Wet cake was added in solution containing azure-B (3.0 gm), process water (1.8 lit), zinc chloride (825 mg) and tetrahydrofuran (3.6 lit) into RBF at 25° C. to 30° C. Reaction mass heated to get 63° C. to 68° C. and stirred for 1.0 hr at 63° C. to 68° C. Reaction mass cooled to 25° C. to 35° C. and maintained for 12.0 hrs. at 25° C. to 30°. Conc. hydrochloric acid (600 mL) was slowly added into the reaction mass at 25° C. to 35° C. and stirred for 3.0 hrs. at 25° C. to 35° C. Reaction mass filtered and washed with fine filtered tetrahydrofuran (300 ml). Material was dried in vacuum oven (with 200 mbar Vacuum) at 30° C. to 35° C. for 2.0 hrs. to obtain Dry wt.=135 gm. and Yield: 90%.

[0094] While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art, from a reading of the disclosure, that various changes in form and detail can be made without departing from the true scope of the invention.