Patent Description:
There is already a wide variety of patents and publications on this topic. Most of them however focus on biologic activity. Carglumic acid and its biologic activity was first described in <NUM> in the publication of <NPL>. Preparation of its deuterated analog was later described in the <NPL>, where a methodology for a similar substance was used, which had been previously described in article <NPL>. None of these works however dealt with purification of the raw product and its detailed analysis.

The synthesis is described in the newer document <CIT>. Reaction of cyanate with glutamic acid in water with pH <NUM>-<NUM> is used according to the scheme <NUM>.

The purity of the final product is <NUM>%. 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 <CIT> Repeated precipitation of the product by sulfuric acid and isolation of a highly pure product <NUM>% is also described in the document <CIT>. A very similar procedure, with a more accurate temperature profile, is also described in the patent <CIT>. 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 <CIT> or <CIT>. The maximum purity achieved through these procedures is <NUM>%. Such products are however unsellable, because the acceptable impurity level can reach maximally <NUM>% 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.

The determined goal is achieved through the invention of a method for obtaining carglumic acid, compound with formula I,
<CHM>
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 acid, or a mixture of water, an organic acid and a mineral acid. An advantage is pH of reaction mixture in the range from <NUM> to <NUM>. The solvate is characterized with NMR according to the enclosure <NUM> and <NUM> and an X-ray powder diffractogram characterized according to the enclosure <NUM>. Raw carglumic acid with formula I was prepared through the reaction of sodium salt of glutamic acid with potassium cyanate according to the scheme <NUM>.

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 <NUM> to <NUM> is used as a precipitating 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, <NUM>-propanol, <NUM>-propanol, acetonitrile, formamide, dimethylformamide, dimethylacetamide, N-methylpyrrolidone or mixtures thereof.

Isolated solvate is preferably used for the preparation of pure carglumic acid. 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 <NUM>, 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, <NUM>-propanol, <NUM>-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 acid or propionic acid or in their solution with water or with organic solvent from the group of methanol, ethanol, <NUM>-propanol, <NUM>-propanol, acetone, acetonitrile, tetrahydrofuran, dimethylformamide, dimethylacetamide, N-methylpyrrolidone.

Another option how to release carglumic acid 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, <NUM>-propanol, <NUM>-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 acid 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, <NUM>-propanol, <NUM>-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 precipitates 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 acid which is solvate of carglumic acid with dimethyl sulfoxide NMR characterized according to the enclosure <NUM> and <NUM> and x-ray powder diffractogram characterized according to the enclosure <NUM> with maximal at <NUM>; <NUM>; <NUM>; <NUM>; <NUM>; <NUM>; <NUM>; <NUM>; <NUM>; <NUM>; <NUM>; <NUM>; <NUM>; <NUM>; <NUM>; <NUM>; <NUM>; <NUM>; <NUM>; <NUM>; <NUM>; <NUM>; <NUM> ° 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.

Into a three necked flask <NUM> equipped with a magnetic stirrer, a thermometer and an air cooler was weighted a monohydrate of monosodium glutamate (<NUM>, <NUM> mmol), measured water (<NUM>) and weighted potassium cyanate (<NUM>, <NUM> mmol). The suspension was stirred and heated to <NUM> and this temperature was kept for a period of <NUM> hour. Afterwards, this clear solution was cooled to <NUM> and it was dropwise acidified with concentrated hydrochloric acid (app. <NUM>) ensuring the temperature would not exceed <NUM>. The suspension was filtrated at this temperature. A filter cake was properly rinsed with ice cold water (<NUM>×<NUM>), acetone (<NUM>×<NUM>) at room temperature and dried in vacuum at <NUM>. The weight of the raw carglumic acid (white powder) after drying: <NUM> (<NUM> %), with HPLC purity <NUM>%.

Into a <NUM> three necked flask equipped with a stirrer, a thermometer, a reflux condenser and dropping funnel was weighted raw product from the example <NUM> (<NUM>) and added dimethyl sulfoxide (<NUM>). The suspension was heated to <NUM> until the solid dissolved; the heating was shut down, cooling water was passed through the reflux condenser and acetone (<NUM>) was dropped through the dropping funnel into the solution during the period of ten minutes. The formed suspension was cooled to <NUM>, solid material was filtrated off; filter cake was rinsed with acetone (<NUM>×<NUM>) at room temperature and dried in vacuum in <NUM>. In this way <NUM> 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 <NUM>-<NUM>.

The crude product from Example <NUM> was treated in the same manner as in Example <NUM>, except that Et<NUM>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 <NUM>.

The crude product from Example <NUM> was treated in the same manner as in Example <NUM>, 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 <NUM>.

The crude product from Example <NUM> was treated in the same manner as in Example <NUM>, 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 <NUM>.

The crude product from Example <NUM> was treated in the same manner as in Example <NUM>, 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 <NUM>.

The crude product from Example <NUM> was treated in the same manner as in Example <NUM>, 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 <NUM>.

The crude product from Example <NUM> was treated in the same manner as in Example <NUM>, 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 <NUM>.

The crystals of the carglumic acid solvate with dimethyl sulfoxide (<NUM>) were dissolved in a hot mixture of distilled water (<NUM>) and acetone (<NUM>). Charcoal (<NUM>) was added to the solution and after five minutes the solution was filtered through a glass filter. The filtrate was cooled to <NUM> 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 <NUM> and then it was cooled with water and ice to <NUM>-<NUM>. After stirring for <NUM> hour at this temperature, the suspension was filtered and washed with ice-cold distilled water (<NUM>×<NUM>) and room temperature acetone (<NUM> × <NUM>). The yield of <NUM> of carglumic acid of formula <NUM> were received, corresponding to a total yield of <NUM>%. HPLC purity was <NUM>%, m. <NUM>-<NUM>.

The solvate of carglumic acid with dimethyl sulfoxide from the example <NUM>, was processed in the same manner as in the Example <NUM> 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 <NUM> and HPLC purity.

Crystalline solvate of carglumic acid with dimethyl sulfoxide prepared according to the Example <NUM> (<NUM>) was stirred up in demineralized water (<NUM>) at a temperature of <NUM>. Quantitative desolvation occurred within <NUM> hours. The solid was filtered off; the filter cake was rinsed with <NUM>% aqueous acetone and the solid was dried in vacuum. The total yield of carglumic acid formula <NUM> on initial monosodium glutamate was <NUM> %, HPLC purity <NUM> %.

Crystalline solvate of carglumic acid with dimethyl sulfoxide prepared according to the example <NUM> (<NUM>) was dissolved in demineralized water (<NUM>) at a temperature of <NUM>. Crystalline carglumic acid precipitated by spontaneous cooling to room temperature. The suspension was filtered, the filter cake was washed with <NUM>% aqueous acetone and the solid was dried in vacuum. The total yield of carglumic acid of formula <NUM> per starting sodium glutamate was <NUM>%, HPLC purity <NUM>%.

The crystalline carglumic acid solvate with dimethyl sulfoxide prepared according to Example <NUM> (<NUM>) was dissolved in demineralized water (<NUM>) at <NUM>. The solution was filtered at <NUM> with activated carbon CX (<NUM>). 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 <NUM> and the HPLC purity.

The crystalline carglumic acid solvate with dimethyl sulfoxide prepared according to Example <NUM> (<NUM>) was suspended in demineralized water (<NUM>) at <NUM>. The pH of the suspension was adjusted to <NUM> with hydrochloric acid (<NUM>%). The suspension was heated to <NUM> and the resulting solution was filtered with CX activated carbon (<NUM>). Crystalline carglumic acid precipitated upon spontaneous cooling to room temperature. The suspension was filtered, the filter cake was washed with <NUM>% aqueous acetone and the solid was dried in vacuum. The total yield of carglumic acid of formula <NUM> per starting sodium glutamate was <NUM>%, HPLC purity <NUM>%.

The crystalline carglumic acid solvate with dimethyl sulfoxide prepared according to Example <NUM> (<NUM>) was suspended in demineralized water (<NUM>) at <NUM>. The pH of the suspension was adjusted to <NUM> with hydrochloric acid (<NUM>%). The suspension was heated to <NUM> and the resulting solution was filtered with charcoal CX (<NUM>). Upon spontaneous cooling to room temperature crystalline carglumic acid precipitated. The suspension was filtered, the filter cake was washed with <NUM>% aqueous acetone and the solid was dried in vacuum. The total yield of carglumic acid of formula <NUM> per starting sodium glutamate was <NUM>%, HPLC purity <NUM>%.

The crystalline carglumic acid solvate with dimethyl sulfoxide prepared according to Example <NUM> (<NUM>) was dissolved in a mixture of demineralized water (<NUM>) and concentrated acetic acid (<NUM>) at <NUM>. The solution was filtered at <NUM> with activated carbon CX (<NUM>). Upon spontaneous cooling to room temperature crystalline carglumic acid precipitated. The suspension was filtered, the filter cake was washed with <NUM>% aqueous acetone and the solid was dried in vacuum. The overall yield of carglumic acid of formula <NUM> per starting sodium glutamate was <NUM>%, HPLC purity <NUM>%.

The crystalline carglumic acid solvate with dimethyl sulfoxide prepared according to Example <NUM> (<NUM>) was stirred in concentrated acetic acid (<NUM>) at <NUM>. Quantitative desolvation occurred within <NUM> 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 <NUM> per starting sodium glutamate was <NUM>%, HPLC purity <NUM>%.

The crystalline dimethyl sulfoxide solvate of carglumic acid prepared according to Example <NUM> (<NUM>) was dissolved in concentrated acetic acid (<NUM>) at <NUM>. The solution was filtered at <NUM> with activated carbon CX (<NUM>), the filtrate was cooled to <NUM> 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 <NUM> per starting sodium glutamate was <NUM>%, HPLC purity <NUM>%.

The crystalline dimethyl sulfoxide solvate of carglumic acid prepared according to Example <NUM> (<NUM>) was suspended at <NUM> in a mixture of concentrated acetic acid (<NUM>) and <NUM>% aqueous hydrochloric acid (<NUM>). Quantitative desolvation occurred within <NUM> 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 <NUM> per starting sodium glutamate was <NUM>%, HPLC purity <NUM>%.

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

The crystalline dimethyl sulfoxide solvate of carglumic acid prepared according to Example <NUM> (<NUM>) was suspended in a mixture of acetone (<NUM>) and <NUM>% aqueous hydrochloric acid (<NUM>) at <NUM>° C. Quantitative desolvation occurred within <NUM> 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 <NUM> per starting sodium glutamate was <NUM>%, HPLC purity <NUM>%.

The crystalline dimethyl sulfoxide solvate of carglumic acid prepared according to Example <NUM> (<NUM>) was dissolved in water (<NUM>) at <NUM>, and acetone (<NUM>) was added to the resulting solution. The solution was filtered with charcoal at <NUM> through a pad of Celite. Free cooling to room temperature precipitated needle-like crystals, which were filtered off and washed with <NUM> x <NUM> of ice water and <NUM> x <NUM> of acetone. The obtained product was dried at <NUM>. The yield <NUM> of carglumic acid of formula <NUM> were obtained with an HPLC purity of <NUM>%.

Claim 1:
Method of purification of carglumic acid, substance formula I,
<CHM>
wherein, it contains the following steps
a) Dissolution of carglumic acid in dimethyl sulfoxide
b) Addition of a precipitating solvent
c) Isolation of the precipitated carglumic acid solvate with dimethyl sulfoxide,
d) Release of carglumic acid from the solvate through treatment with water, optionally a mixture of water and an organic solvent, optionally a mixture of water and an organic or mineral acid, optionally a mixture of water, organic acid and mineral acid, optionally with an organic acid, optionally with a mixture of water, mineral acid and organic solvent.