Field of the Invetion

The presented invention belongs into the field of organic chemistry and it describes a method of purification of cargiumic acid and an intermediate of this method.

Prior Art

There is already a wide variety of patents and publications on this topic. Most of them however focus on biologic activity. Cargiumic acid and its biologic activity was first described in 1952 in the publication of Griso!ia S. et al.: J. Biological Chem. 1952, 198, 561-71. Preparation of its deuterated analog was later described in the Griso!ia S. et al.: J. Biological Cham, 1954, 210, 109-17, where a methodology for a similar substance was used, which had been previously described in artide Nyc J. et al.: J. Am. Chem. Soc. 1947, 69, 1382. None of these works however dealt with purification of the raw product and its detailed analysis.

The synthesis is described in the newer document CN108484448 A. Reaction of cyanate with glutamic acid in water with pH 11-13 is used according to the scheme 1.

Scheme 1

The purity of the final product is 92%. The product is purified through crystallizations and precipitation as in the previous case. The purification through repeated precipitation and crystallizations is also described in the patent CN 108017561 A. Repeated precipitation of the product by sulfuric acid and isolation of a highly pure product 99% is also described in the document CN 107033035 A. A very similar procedure, with a more accurate temperature profile, is also described in the patents reactor is then also described in another patent CN 103980163 A. It however does not deal with purification of the product. Preparation of the compound was described many decades ago. In the current times, essentially the same methods are used, the descriptions are only more detailed. The older works did not deal with the purity of the substance at all, while the newer works described only repeated precipitation, use of various acids and influences of pH, which was described in the patents CN 107033035 A or CN 105601542 A. The maximum purity achieved through these procedures is 99%. Such products are however unsellable, because the acceptable impurity level can reach maximally 0.1% weight percentage. The required higher purity is reached with repetition of the mentioned procedure. This is a time and money consuming process, and moreover, it causes the amount of waste to increase, while the yield decreases.

The aim of the presented invention is to introduce a procedure of purification of carglumic acid - formula I - to high purity through a simple and cheap procedure.

Summary of the invention

The determined goal is achieved through the invention of a method for obtaining carglumic acid, compound with formula I,

I when the raw carglumic acid is dissolved in dimethyl sulfoxide and solvate of carglumic acid and dimethyl sulfoxide is formed. A solvent, in which the solvate of carglumic acid with dimethyl sulfoxide is insoluble, is then added to the generated solution, thereby precipitating the solvate of carglumic acid with dimethyl sulfoxide. The precipitated solvate of carglumic acid with dimethyl sulfoxide is isolated and carglumic acid is subsequently released from it by treatment with water or a mixture of water and an organic or mineral add, or a mixture of water, an organic add and a mineral acid. An advantage is pH of reaction mixture in the range from 0 to 5.5. The solvate is characterized with NMR according to the endosure 1 and 2 and an X-ray powder d ^' rffractogram characterized according to the endosure 3. Raw carglumic acid with formula I was prepared through the reaction of sodium salt of glutamic acid with potassium cyanate according to the scheme 2.

Scheme 2

The solvate of carglumic acid with dimethyl sulfoxide is preferably isolated in cold and acetonitrile or a neutral organic solvent with relative permittivity in the range from 2.1 to 29 is used as a predpitating solvent.

The release of carglumic acid from its solvate with dimethyl sulfoxide is preferably carried out with hot water with the addition of a solvent selected from the group of acetone, tetrahydrofuran, methanol, ethanol, 1 -propanol, 2-propanol, acetonitrile, formamide, dimethylformamide, dimethylacetamide, N-methylpyrrolidone or mixtures thereof. isolated solvate is preferably used for the preparation of pure carglumic add. Various combinations can be considered. One from the options for solvate decomposition is to use water itself. It is optimal if the desolvation of the solvate takes place at elevated temperatures, ideally when all the crystalline material is dissolved, but the reaction also takes place at temperatures below 10°C, but it takes many times more time. It is possible to use water with organic or mineral acid instead of water. If the decomposition of the solvate does not take place under optimal conditions, carglumic acid of the formula I can be purified through crystallization from water preferably with presence of a solvent. A suitable solvent can be for example: methanol, ethanol, 2-propanol, 1 -propanol, acetone, acetonitrile, tetrahydrofuran, dimethylformamide, dimethylacetamide, N-methylpyrrolidone, dimethyl sulfoxide.

Another way to release carglumic acid of formula I from its solvate with DMSO is to stir or dissolve the solvate at elevated temperature in an organic acid which can be for example formic acid, acetic add or propionic add or in their solution with water or with organic solvent from the group of methanol, ethanol, 2-propanol, 1 -propanol, acetone, acetonitrile, tetrahydrofuran, dimethylformamide, dimethylacetamide, N- methylpyrrolidone.

Another option how to release carglumic add of formula I from its solvate with dimethyl sulfoxide is through stirring or crystallization at elevated temperature in organic solvent from the group - methanol, ethanol, 2-propanol, 1 -propanol, acetone, acetonitrile, tetrahydrofuran, dimethylformamide, dimethylacetamide, N- methylpyrrolidone, dimethyl sulfoxide with addition of water solution of mineral acid.

Particularly preferable is when the solvate of carglumic acjd with dimethyl sulfoxide is dissolved in water at higher temperatures, possibly in a mixture of water with an organic solvent, which can be selected from the group - methanol, ethanol, 2- propanol, 1 -propanol, acetone, acetonitrile. The solution can be further purified by filtration with charcoal, kieselguhr or another sorbent After cooling, pure carglumic acid of the formula I predpitates out of the solution. It is then separated from the reaction mixture, for example by filtration, it is washed with water or a mixture of water with solvents or anhydrous solvents and it is dried.

The subject of the invention is likewise an intermediate of the above mentioned method of purification of carglumic add which is solvate of carglumic add with dimethyl sulfoxide NMR characterized according to the enclosure 1 and 2 and x-ray powder diffractogram characterized according to the enclosure 3 with maximal at 36.088; 35.309; 34.866; 31.464; 30.129; 28.865; 27.913; 27.44; 25.838; 24.427; 24.216; 23.654; 22.478; 21.88; 20.607; 19.328; 18.855; 18.562; 17.812; 17.399; 14.929; 11.593; 7.44 ° two-theta. The presented invention achieves newer and higher efficiency, since the carglumic acid solvate with dimethyl sulfoxide is sparingly soluble in mixtures of dimethyl sulfoxide with above mentioned solvents, but the impurities and concomitant substances which remain in the mother liquors are very soluble in them. The solvate can be further dissolved in water, or mixtures thereof with the above-mentioned solvents and acids, and at this stage it can be further purified by filtration with a suitable absorbent such as charcoal. The use of a carglumic acid solvate with dimethyl sulfoxide is advantageous, mainly due to the simplicity of processing, high yields and cleaning power.

The examples of particular implementations are described below, they do not in any way limit the extent of the protection described in the definition, they solely provide clarification of the disclosure of the invention.

Enclosure description

Enclosure 1 is ¹ H-NMR of the solvate of carglumic acid with dimethyl sulfoxide

Enclosure 2 is ¹³ C-NMR of the solvate of carglumic acid with dimethyl sulfoxide

Enclosure 3 is an x ray powder diffractogram of the solvate of carglumic acid with dimethyl sulfoxide

Examples

Example 1 : Preparation of raw carglumic acid

Into a three necked flask 250 mL equipped with a magnetic stirrer, a thermometer and an air cooler was weighted a monohydrate of monosodium glutamate (37.4 g, 200 mmol), measured water (100 mL) and weighted potassium cyanate (20 g, 246.5 mmol). The suspension was stirred and heated to 70 °C and this temperature was kept for a period of 1 hour. Afterwards, this clear solution was cooled to 0°C and it was dropwise acidified with concentrated hydrochloric acid (app. 38 mL) ensuring the temperature would not exceed 3°C. The suspension was filtrated at this temperature. A filter cake was properly rinsed with ice cold water (3x50 mL ), acetone (3x30 mL) at room temperature and dried in vacuum at 35°C. The weight of the raw carglumic acid (white powder) after drying: 28.5 g (75 %), with HPLC purity 95%.

Example 2: Preparation of solvate of carglumic acid

Into a 250 mL three necked flask equipped with a stirrer, a thermometer, a reflux condenser and dropping funnel was weighted raw product from the example 1 (28.5 g) and added dimethyl sulfoxide (42 mL). The suspension was heated to 90 °C until the solid dissolved; the heating was shut down, cooling water was passed through the reflux condenser and acetone (185 mL) was dropped through the dropping funnel into the solution during the period of ten minutes. The formed suspension was cooled to 5 °C, solid material was filtrated off; filter cake was rinsed with acetone (3x30 mL) at room temperature and dried in vacuum in 35 °C. In this way 35.6 g of white crystalline material was received. According to the NMR and HPLC analysis, it is a solvate of carglumic acid with dimethyl sulfoxide. Melting point of the solvate is 112-114 °C.

Example 3: Preparation of solvate of carglumic acid

The crude product from Example 1 was treated in the same manner as in Example 2, except that Et ₂ O was added to the solution instead of acetone. A white crystalline solvate of carglumic acid with dimethyl sulfoxide was obtained in a yield of 33.5 g.

Example 4: Preparation of solvate of carglumic acid

The crude product from Example 1 was treated in the same manner as in Example 2, except that ethyl acetate was added to the solution instead of acetone. A white crystalline solvate of carglumic acid with dimethyl sulfoxide was obtained in a yield of 36.2 g.

Example 5: Preparation of solvate of carglumic acid The crude product from Example 1 was treated in the same manner as in Example 2, except that toluene was added to the solution instead of acetone. A white crystalline solvate of carglumic acid with dimethyl sulfoxide was obtained in a yield of 36 g.

Example 6: Preparation of solvate of carglumic acid

The crude product from Example 1 was treated in the same manner as in Example 2, except that cyclohexanone was added to the solution instead of acetone. A white crystalline solvate of carglumic acid with dimethyl sulfoxide was obtained in a yield of 33 g.

Example 7: Preparation of solvate of carglumic acid

The crude product from Example 1 was treated in the same manner as in Example 2, except that acetonitrile was added to the solution instead of acetone. A white crystalline solvate of carglumic acid with dimethyl sulfoxide was obtained in a yield of 34 g.

Example 8: Preparation of solvate of carglumic acid

The crude product from Example 1 was treated in the same manner as in Example 2, except that dichloroethane was added to the solution instead of acetone. A white crystalline solvate of carglumic acid with dimethyl sulfoxide was obtained in a yield of 36 g.

Eample 9: Desolvation

The crystals of the carglumic acid solvate with dimethyl sulfoxide (35.6 g) were dissolved in a hot mixture of distilled water (106 mL) and acetone (10.8 mL). Charcoal (1.2 g) was added to the solution and after five minutes the solution was filtered through a glass filter. The filtrate was cooled to 40°C and seeded with stirring off. As the needle crystals grew, gentle agitation was started. The temperature of the suspension was allowed to drop spontaneously to 25°C and then it was cooled with water and ice to 0-5°C. After stirring for 1 hour at this temperature, the suspension was filtered and washed with ice-cold distilled water (3x20 mL) and room temperature acetone (3 x 30 mL). The yield of 21.3 g of carglumic acid of formula 1 were received, corresponding to a total yield of 56%. HPLC purity was 99.97%, m.p. 176-178°C.

Examples 10 - 19: Desolvation

The solvate of carglumic acid with dimethyl sulfoxide from the example 2, was processed in the same manner as in the Example 9 except that a solvent was added to the reaction mixture according to the following table, which shows the total yield of carglumic acid of formula 1 and HPLC purity.

Example 20: Stirring in water

Crystalline solvate of carglumic acid with dimethyl sulfoxide prepared according to the Example 2 (20 g) was stirred up in demineralized water (60 mL) at a temperature of 20 °C. Quantitative desolvation occurred within 24 hours. The solid was filtered off; the filter cake was rinsed with 10% aqueous acetone and the solid was dried in vacuum. The total yield of carglumic acid formula 1 on initial monosodium glutamate was 59 %, HPLC purity 99.2 %.

Example 21: Crystallization from water Crystalline solvate of carglumic acid with dimethyl sulfoxide prepared according to the example 2 (20 g) was dissolved in demineralized water (60 mL) at a temperature of 90°C. Crystalline carglumic acid precipitated by spontaneous cooling to room temperature. The suspension was filtered, the filter cake was washed with 10% aqueous acetone and the solid was dried in vacuum. The total yield of carglumic acid of formula 1 per starting sodium glutamate was 46%, HPLC purity 99.57%.

Examples 22 - 31: Crystallization from water with addition of solvent

The crystalline carglumic acid solvate with dimethyl sulfoxide prepared according to Example 2 (20 g) was dissolved in demineralized water (60 mL) at 90°C. The solution was filtered at 90°C with activated carbon CX (1g). Crystalline carglumic acid precipitated after spontaneous cooling to room temperature. The solvent listed in the following table was added to the precipitated carglumic acid suspension. It also shows the total yield of carglumic acid of formula 1 and the HPLC purity.

Example 32: Crystallization from water and mineral acid

The crystalline carglumic acid solvate with dimethyl sulfoxide prepared according to Example 2 (20 g) was suspended in demineralized water (60 mL) at 20°C. The pH of the suspension was adjusted to 0 with hydrochloric acid (35%). The suspension was heated to 90°C and the resulting solution was filtered with CX activated carbon (1 g). Crystalline carglumic acid precipitated upon spontaneous cooling to room temperature. The suspension was filtered, the filter cake was washed with 10% aqueous acetone and the solid was dried in vacuum. The total yield of carglumic acid of formula 1 per starting sodium glutamate was 43%, HPLC purity 99.12%.

Example 33: Crystallization from water and mineral acid

The crystalline carglumic acid solvate with dimethyl sulfoxide prepared according to Example 2 (20 g) was suspended in demineralized water (60 mL) at 20°C. The pH of the suspension was adjusted to 0.5 with hydrochloric acid (35%). The suspension was heated to 90°C and the resulting solution was filtered with charcoal CX (1 g). Upon spontaneous cooling to room temperature crystalline carglumic acid precipitated. The suspension was filtered, the filter cake was washed with 10% aqueous acetone and the solid was dried in vacuum. The total yield of carglumic acid of formula 1 per starting sodium glutamate was 45%, HPLC purity 99.41%.

Example 34: Crystallization from water and organic acid The crystalline carglumic acid solvate with dimethyl sulfoxide prepared according to Example 2 (20 g) was dissolved in a mixture of demineralized water (60 mL) and concentrated acetic acid (5 mL) at 90°C. The solution was filtered at 90°C with activated carbon CX (1 g). Upon spontaneous cooling to room temperature crystalline carglumic acid precipitated. The suspension was filtered, the filter cake was washed with 10% aqueous acetone and the solid was dried in vacuum. The overall yield of carglumic acid of formula 1 per starting sodium glutamate was 47%, HPLC purity 99.5%.

Example 35: Stirring in organic acid

The crystalline carglumic acid solvate with dimethyl sulfoxide prepared according to Example 2 (20 g) was stirred in concentrated acetic acid (100 mL) at 90°C. Quantitative desolvation occurred within 1 hour. The suspension was filtered, the filter cake was washed with acetone and the solid was dried in vacuo. The overall yield of carglumic acid of formula 1 per starting sodium glutamate was 50%, HPLC purity 99.50%.

Example 36: Crystallization from organic acid

The crystalline dimethyl sulfoxide solvate of carglumic acid prepared according to Example 2 (20 g) was dissolved in concentrated acetic acid (300 mL) at 120°C. The solution was filtered at 120°C with activated carbon CX (1 g), the filtrate was cooled to 60°C and the solution was seeded with a crystal of carglumic acid. Upon spontaneous cooling to room temperature crystalline carglumic acid precipitated. The suspension was filtered, the filter cake was washed with acetone and the solid was dried in vacuum. The total yield of carglumic acid of formula 1 per starting sodium glutamate was 40%, HPLC purity 99.48%.

Example 37: Stirring up In organic acid and water solution on mineral acid The crystalline dimethyl sulfoxide solvate of carglumic acid prepared according to Example 2 (20 g) was suspended at 20°C in a mixture of concentrated acetic acid (200 mL) and 3% aqueous hydrochloric acid (6 ml_). Quantitative desolvation occurred within 1 hour. The suspension was filtered, the filter cake was washed with acetone and the solid was dried in vacuum. The overall yield of carglumic acid of formula 1 per starting sodium glutamate was 55%, HPLC purity 98.93%.

Example 38: Crystallization from water solution of mineral acid with solvent

The dimethyl sulfoxide solvate of carglumic acid from Example 2 was treated in the same manner as in Example 9, except that 3% aqueous hydrochloric acid was added instead of water to dissolve the solvate. The overall yield of carglumic acid of formula 1 per starting sodium glutamate was 53%, HPLC purity 98.57%.

Example 39: Stirring up in mixture water, acetone, mineral acid The crystalline dimethyl sulfoxide solvate of carglumic acid prepared according to Example 2 (20 g) was suspended in a mixture of acetone (60 mL) and 3% aqueous hydrochloric acid (6 mL) at 20° C. Quantitative desolvation occurred within 24 hours. The suspension was filtered, the filter cake was washed with acetone and the solid was dried in vacuum. The overall yield of carglumic acid of formula 1 per starting sodium glutamate was 57%, HPLC purity 98.97%.

Example 40: Re-purification of polluted carglumic acid

The crystalline dimethyl sulfoxide solvate of carglumic acid prepared according to Example 24 (5 g) was dissolved in water (20 mL) at 90°C, and acetone (2 ml_) was added to the resulting solution. The solution was filtered with charcoal at 50°C through a pad of Celite. Free cooling to room temperature precipitated needle-like crystals, which were filtered off and washed with 3 x 2 mL of ice water and 3 x 2 mL of acetone. The obtained product was dried at 40°C. The yield 4 g of carglumic acid of formula 1 were obtained with an HPLC purity of 99.97%.

Industrial applicability

The invention is useful in the pharmaceutical industry in preparation of important medicaments based on carglumic acid.