RU2109015C1 - Method for producing folic acid derivative (6s)-isomers - Google Patents

Abstract

FIELD: medical industry; production of vitamins. SUBSTANCE: commercial-scale method for production of folic acid derivative (6S)-isomers is based on chromatographic separation of mixture consisting of two (6RS)-diastereoiromers on chromatographic column using as separating agent, albumin dissolved in buffer solution having pH=5. Solution of mixture of (6RS)-diastereoisomers has concentration 0.1-10 wt.-%. EFFECT: higher efficiency. 8 cl

Description

 The invention relates to a method for the industrial production of (6S) -folic acid derivatives by chromatographic separation of a mixture of diastereoisomers on a column, in particular a method for the industrial production of 5- (methyl) - (6S) -tetrahydrofolate acid and 5- (formyl) - (6S) -tetrahydrofolate acid marked with "MTHF" and "FTHF". MTHF and FTHF are physiological molecules that, together with the less stable 6,10-methylenetetrahydrofolic acid and tetrahydrofolic acid, form in vivo active forms of folic acid, commonly called folic acid cofactors.

 From a therapeutic point of view, both MTHF and FTHF are used in all cases of folate deficiency, usually to protect the liver, less often in antitumor therapy.

где
X = CH₃ или CHO;
а R₁ и R₂ являются H, NH $\genfrac{}{}{0ex}{}{\text{+}}{\text{4}}$ , щелочными или щелочноземельными металлами, причем R₁ и R₂ могут быть одинаковыми или отличаться друг от друга.MTHF, FTHF, and therapeutically useful derivatives commonly used, are represented by the following formula (1):

Where
X is CH ₃ or CHO;
and R ₁ and R ₂ are H, NH $\genfrac{}{}{0ex}{}{\text{+}}{\text{4}}$ alkali or alkaline earth metals, wherein R ₁ and R ₂ may be the same or different from each other.

 The compounds of formula (1) have two asymmetric hydrocarbon atoms and, therefore, can exist in four diastereoisomeric forms.

 It is well known that usually when stereoisomeric pharmaceutical forms exist, only one of them is active, the rest are inactive or even harmful. In this case, it is well known that the (6S) forms are active.

 Some studies have been carried out regarding the industrial production and / or isolation of two diastereoisomers and the direct synthesis of the (6S) form.

 Stereospecific analysis of the (6S) -form (both MTHF and FTHF) as well as the separation of two (6RS) -diastereoisomers is difficult, resulting in structurally unstable final products.

 The synthesis of a stereoisomeric mixture of MTHF and FTHF is described in GB-A-1 572138 and CH-A-496012, respectively.

 In order to isolate (6S) -diastereoisomers from mixtures thus obtained, it is possible to transfer the mixture of stereoisomers to the corresponding 5-methoxycarbonyl derivatives or to other derivatives with a chiral center: thus using different solubilities in appropriate solvents, it is possible to separate the two indicated diastereoisomers, (6R) and (6S) [2]. The specified method is complicated, and the yield is extremely low.

 Other separation methods are based on multiple crystallizations of both the (6R, S) mixture [1] and the intermediate, but always give low yields.

 The last separation of mixtures of stereoisomers of MTHF and FTHF was obtained in [3] by fractional crystallization and hydrolysis or reduction (optionally) of a 5,10-methylene- (6RS) -tetrahydrofolium intermediate, which is very difficult to operate and isolate from a harmful and toxic solvent, such like formic acid.

 A method for isolating the (6S) form is disclosed in [1], in which a solution of a calcium or magnesium salt of said mixture of stereoisomers is treated with an amine, precipitating a salt of the specified cation as an oxalate, and obtaining the desired product by fractional crystallization and reverse conversion to a calcium or magnesium salt.

 The resulting yields are quite low, and the method involves several stages of obtaining salts and crystallization.

 In another separation method, the racemic mixture in the form of a calcium salt (EP 367902) is added to a certain amount of organic and inorganic salts, in particular sodium iodide, thereby obtaining a product with a high degree of purity, but again with a sufficiently low yield, which is undoubtedly senseless with industrial points of view.

 Another method for the separation of FTHF isomers [4] is known, according to which silica gel impregnated with bovine serum albumin (BSA) is used. A phosphate buffer with a pH range from 6.4 to 8.4, preferably 7.4, is used as eluant.

 The eluate obtained in accordance with the method of Choi et al. Contains a very small amount of isomer (6S), suitable exclusively for analytical purposes.

 The application of the method of Choi et al. Under industrial conditions would result in a rather large delay time of the solution in the column due to poor separation, which is not suitable for industrial production, since to obtain a suitable separation, it would be necessary to use the column for an extremely long time or act as a solvent on one column several times, which increases the possibility of separation of the mixture and greatly increases the consumption of reagents.

 It was found that in accordance with the invention, following the methodology of Choi et al., But using a buffer solution with a very narrow pH range (between 5.0 and 5.5) as an elution mixture (6R, S), isolation (6S ) - isomers with an industrial yield are cheaper and simpler.

 Using two industrial columns filled with silica gel impregnated with albumin and separating the (6R, S) mixture in accordance with the method of Choi et al. And the method of the invention, yields of the (6S) isomer are lower than 5% and not lower than 20%, respectively.

 In addition to the methods described above for the isolation of (6S) isomers from a mixture of (6S, R), attempts have been made to direct synthesis of (6S) isomers.

 According to EP-A-356984, the synthesis of the (6S) -isomer was carried out by a method based on the stereospecific hydrogenation of the (5-6) double bond of 7.8-dihydrofolate. If the indicated hydrogenation is carried out by dihydrofoliate reductase in the presence of NADP, good conversion is achieved, however, this method is extremely complicated and not always suitable from an industrial point of view.

 Other types of stereospecific hydrogenation have been tried using suitable catalysts, but the results have always been poor (Tetrahedron 42, 117, 1986).

 Finally, today there is no cheap method suitable industrially for producing the (6S) -isomers of MTHF and FTHF. As for the practical therapeutic use, (6S) -MTHF and (6S) -FTHF are not used as such, but are used as salts, usually calcium salts. Such a conversion is carried out by well-known methods, for example, in accordance with the methods disclosed in US Pat. No. 2,888,018 and Weast "Handlook of Chemistry and Physics (57th Edition: p. B-100).

 From the mixture of (6R, S) -MTHF or (6R, S) -FTHF, the corresponding (6S) -isomer is isolated by the method in which an aqueous albumin solution is loaded into the chromatographic column, then washed with the first buffer solution, an aqueous solution of a mixture of diastereoisomers of the derivative is loaded of formula (1), then elute with a second buffer solution, obtaining (6S) -diastereoisomer as the first eluate for FTHF and the second eluate for MTHF, the method is characterized in that the concentration of the albumin solution is from 0.1 to 10 wt.%, the solution of derivatives has concentration from 0.1 to 10 m wt.%, and the pH of these buffer solutions is 4.8 to 5.8.

 It is preferable to first rinse the column with a pH 7 buffer solution and then with a pH of 5 before loading the mixture of diastereoisomers of the derivative of formula (1).

 It is further preferred that the pH of aqueous solutions of albumin and a mixture of diastereoisomers is maintained in the range of 4.8-5.8. In addition, it is preferable that the silica gel have a particle size distribution (granule size) between 25 and 60 μm, more preferably 35 to 45 μm.

 The preferred buffer solutions are phosphate buffers with a concentration of 0.05 - 0.15 M, the concentration of the specified albumin in its aqueous solution is about 1 wt.%, And the concentration of the mixture of diastereoisomers in the solution is about 5 wt.%, While albumin chooses from the group comprising pork, bovine, sheep, rabbit, chicken and egg albumin.

 It is obvious that the buffer solutions used in different phases of the proposed method may be the same or different from each other. As buffer solutions, solutions of phthalates, phosphates, acetates, etc., preferably phosphates, can be used.

 According to a preferred method, 0.05 M phosphate buffer (0.15 M phosphate buffer in the case of calcium salt (6R, S) is used as an eluent for a chromatographic column preloaded with a solution of calcium salt of (6R, S) -methyl-tetrahydrofolate acid) ) -formyl-tetrahydrofolate acid).

 Finally, in accordance with the invention, it is possible to bring the performance of the division method to an industrial level, achieving an extremely short elution time.

 The invention is described in detail with reference to preferred options. However, it is in no way limited to these individual options.

 The outputs presented in the following examples are expressed as weight values, which correspond to a double optical output.

 Example 1. In an industrial column with a diameter of 0.90 m and a height of 6.0 m load silica gel (particle diameter 35/45 μm suspended in a 0.15 M buffer aqueous solution at pH 5 containing mercaptoethanol (0.1 wt.%), filling the column almost completely (to a height of 5.5 m, which corresponds to about 3600 g of silica gel).

 The preparation of a chiral substrate is carried out as follows, a solution of egg serum albumin (1% in 0.15 M phosphate buffer, pH 5.0) is loaded into the column, following absorption in UV at 280 nm on an eluted liquid. The amount of albumin absorbed on silica gel is 200/400 kg. Elution is carried out using 10,000 L of 0.15 M phosphate buffer with a pH of 5.0, then 10,000 L of 0.15 M phosphate buffer with a pH of 7 and finally 10,000 L of 0.15 M phosphate buffer with a pH of 5.0. Then add a 5% solution of the calcium salt of 5-formyl- (6R, S) -tetrahydrofolic acid equivalent to 5 kg (the expiration time is 200/400 l / h), eluting with 0.15 M phosphate buffer with pH 5.0. Fractions of 500 liters are collected.

 (6S) fractions elute before the (6R) fractions. The elution of the fractions can be monitored along the way using a polarimeter equipped with a flow cell, and then accurately metered using high pressure liquid chromatography (HPLC). Sufficiently pure fractions are isolated for the required purposes. Pure fractions are collected and converted into salt by known methods.

 Yield: 1.9 (28%) of (6S) calcium folinate. Title: HPLC> 98%. Optical purity> 97%.

 Example 2. In this example, the method of Choi et al. Is reproduced at an industrial level. Using the same column type and the same method of preparing the stationary phase, the method is carried out as in example 1. Elution is carried out using 10,000 L of 0.15 M phosphate buffer with pH 7 A 5% solution of calcium salt of (6R, S) -folic acid equivalent to about 5 kg is added to the column at a flow rate of 200/400 l / h. Elution is carried out with phosphate buffer with a pH of 7. Collect fractions of 500 l

 Separation unsatisfactory; the purification cycle is repeated four times, trying to obtain a partial separation. Text is discarded due to insufficient separation.

 Example 3. Use the same type of column as in Example 1 and the same method of preparing the stationary phase, but use pig serum albumin (PSA) in this example.

 Yield: 1.5 kg (30%) of (6S) calcium folinate. HPLC titer:> 98%. Optical purity:> 97%.

 Example 4. Use the same type of column as in Example 1, but degassed water is used to prepare the stationary phase and for the subsequent stages of absorption and elution.

 For elution, 10,000 L of 0.15 M phosphate buffer with a pH of 5.2 is used, a 5% solution of (6S) methyl tetrahydrofolate acid, equivalent to 10 kg, is added at a flow rate of 200/400 l / h; again eluting with 0.05 M phosphate buffer at pH 5.2, collecting 500 l fractions.

 (6S) fractions elute after the (6R) fractions. Sufficiently pure fractions are isolated for the required purposes, collected and processed by known methods.

 Yield: 3.9 kg (39%) of the calcium salt of (6S) -methyl tetrahydrofolate. HPLC titer:> 97%. Optical purity:> 97%.

Claims (8)

1. The method of obtaining the (6S) -isomers of folic acid derivatives of General formula I

where X represents CH ₃ or CHO;
R ₁ and R ₂ are H, NH $\genfrac{}{}{0ex}{}{\text{+}}{\text{4}}$ alkali and alkaline earth metals, wherein R ₁ and R ₂ may be the same or different from each other,
by separating a mixture of (6R, S) diastereoisomers of the general formula I, including passing an aqueous albumin solution through a column, washing the column with an aqueous solution of phosphate buffer, loading a mixture of (6R, S) diastereoisomers of the formula I, and eluting (6S) isomers with an aqueous solution of phosphate buffer, characterized the fact that the concentration of albumin in the solution is 0.1 - 10 wt.%, washing the column is carried out in three stages - with a buffer solution with pH 5, then with a buffer solution with pH 7 and, finally, with a buffer solution with pH 5, the concentration of the mixture solution ( 6R, S) di stereoisomers of 0.1 - 10 wt%, a pH buffer solution to elute 4.8 -. 5.8.  2. The method according to claim 1, characterized in that said buffer solutions are phosphate buffers with a concentration between 0.05 and 0.15 M.  3. The method according to claims 1 and 2, characterized in that said column is loaded with silica gel with a granule size of 25-60 μm.  4. The method according to claim 3, characterized in that the size of the silica gel granules is 35 - 45 microns.  5. The method according to PP. 1 to 4, characterized in that the concentration of the specified albumin in its aqueous solution is about 1 wt.%, And the concentration of the specified mixture of diastereoisomers is about 5 wt.%.  6. The method according to claims 1 to 5, characterized in that said albumin is selected from the group comprising pork, bovine, sheep, rabbit, chicken and egg albumin.  7. The method according to claims 1 to 6, characterized in that said aqueous albumin solution is buffered at pH 5.  8. The method according to claims 1 to 6, characterized in that the aqueous solution of the specified mixture of diastereoisomers is buffered at pH 5.3