The method of obtaining (6s)-isomers of the derivatives of folic acid

 

(57) Abstract:

Usage: in health, in particular the vitamin industry. The inventive method for industrial preparation (6 S)-isomers of the derivatives of folic acid by chromatographic separation of the mixture of the two (6 RS)-diasteroisomers on the column, where dividing by the agent is albumin in buffer solution with pH 5, the concentration of the solution mixture (6 R, S)-diastereoisomer of 0.1 - 10 wt.%. 7 C.p. f-crystals,

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

From a therapeutic point of view and MTHF, and FTHF find application in all cases of folate-deficiency as a rule, to protect the liver, less in antitumor terajima (1):

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where

X = CH3or CHO;

and R1and R2are H, NH+4alkaline or alkaline earth metals, and R1and R2can be the same or different from each other.

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

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

Conducted some research on industrial production and/or secretion of the two diastereoisomers and direct synthesis of (6S)-form.

Stereospecific analysis of (6S)-form (and MTHF, and FTHF) as well as the separation of the two (6RS)-diastereoisomers is difficult, which receive structurally unstable end products.

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

To highlight the (6S)-diastereoisomers from mixtures obtained in this way, it is possible to convert a mixture of stereoisomers in sootvetstvuyushiye solubility in appropriate solvents, it is possible to separate these two diastereoisomer, (6R) and (6S) [2]. This method is complex, and the output of this are extremely low.

Other methods of separation based on multiple crystallization as (6R, S) - mixture [1], as well as an intermediate product, but always give a low outputs.

The last separation of mixtures of stereoisomers MTHF and FTHF obtained in [3] fractionated crystallization and hydrolysis or restoring (optional) 5,10-methylene-(6RS)-tetrahydrofolate intermediate product, which is very difficult to operate and to separate from harmful and toxic solvent, such as formic acid.

The allocation method (6S)-form disclosed in [1], in which the salt solution of calcium or magnesium specified mixture of stereoisomers is treated with an amine, precipitating in the form of the oxalate salt of the specified cation and obtaining the desired product by fractionated crystallization and reverse transformation into a salt of calcium or magnesium.

The resulting outputs are low, and the method includes several stages of obtaining salt and crystallization.

In another method of separation of the racemic mixture in the form of calcium salts (EP 367902) is added to a certain amount of organic and reorganize is fairly low output, of course that is useless from the industrial point of view.

Known another method of separating isomers FTHF [4], according to which use silica gel impregnated with bovine serum albumin (BSA). As eluant use phosphate buffer with a range of pH from 6.4 to 8.4, preferably 7,4.

The eluate obtained in accordance with the method of Choi and others, contains a very small amount of the isomer of (6S), usable solely for analytical purposes.

The application of the method Choi, etc. in an industrial environment would result in a large enough delay time of the solution in the column due to weak division that is not suitable for industrial production, because to obtain a suitable separation would need to use a column for an extremely long time, or to act in a solvent on a single column multiple times, which increases the possibility of separation of the mixture and greatly increases the consumption of reagents.

It has been found that in accordance with the invention, following the method of Choi and others , but using as elution of a mixture of (6R,S) buffer solution with a very narrow range of pH values (between 5.0 and 5.5), receive an allocation of (6S) - isomer with industrial output dash is m albumin, and sharing (6R,S) - mixture in accordance with the method of Choi and others and method according to the invention, receives the outputs of the (6S)-isomer below 5% and not below 20%, respectively.

In addition to the above cues (6S)-isomer from a mixture of (6S,R), attempts were made direct synthesis of (6S)-isomer.

In accordance with patent EP-A-356984 synthesis of (6S)-isomer was performed by the method based on the stereospecific hydrogenation (5 - 6)-double bond 7,8-digidrofolieva acid. If the hydrogenation is conducted dihydrofolate reductase in the presence of NADP, achieve good conversion but this method is extremely complex and not always suitable from a manufacturing standpoint.

Has been tested with other types of stereospecific hydrogenation using a suitable catalyst, but the results were always poor (Tetrahedron 42, 117, 1986).

Finally, there is no cheap way that is appropriate in the industrial sense to obtain (6S)-isomers MTHF and FTHF. With regard to the practical therapeutic applications, (6S)-MTHF and (6S)-FTHF not used as such, but are used as salts, usually calcium salts. This transformation is conducted well-known methods, for example, in accordance with the methods, industry (6R,S)-FTHF allocate appropriate (6S)-isomer way in accordance with which in the chromatographic column loaded aqueous solution of albumin, then washed first buffer solution, upload an aqueous solution of a mixture of diastereoisomers derivative of formula (1), then elute the second buffer solution, receiving (6S)-diastereoisomer as the first eluate to FTHF and the second eluate to MTHF method differs in that the concentration of albumin is from 0.1 to 10 wt.%, the solution is derived has a concentration of from 0.1 to 10 wt.%, as specified pH buffer solutions is 4.8 to 5.8.

Preferably first washed with column buffer solution with a pH of 7, and then with a pH of 5 to download mix diastereoisomers derivative of formula (1).

More preferably, the pH of aqueous solutions of albumin and mixtures of diastereoisomers was maintained in the range of 4.8 to 5.8. In addition, it is preferable that the silica gel had a grain size distribution (grain size) between 25 and 60 μm, more preferably 35 to 45 microns.

Preferential buffers are phosphate buffers with a concentration of 0.05 - 0.15 M, the concentration of the specified albumin in its aqueous solution has a value of about 1 wt.%, and the concentration of the mixture of diastereoisomers in solution is ve is egg albumin.

Obviously, buffer solutions used in different phases of the proposed method may be the same or different from each other. As buffer solutions, you can use the solutions of phthalates, phosphates, acetates, etc., preferably phosphates.

In accordance with the preferred method as eluent for chromatographic columns, pre-loaded with a solution of the calcium salt of (6R,S)-methyl-tetrahydrofolate acid, using 0.05 M phosphate buffer (0.15 M phosphate buffer in the case of the calcium salt of (6R,S)-formyl-tetrahydrofolate acid).

Finally, in accordance with the invention, it is possible to increase the performance of the method of division to the industrial level, reaching a very short time afterwards.

The invention is described in detail with reference to preferential options. However, it is in no way limited to those specific options.

The outputs presented in the following examples, expressed in terms of weight values, which corresponds to the dual optical output.

Example 1. In the industrial column with a diameter of 0.90 m and a height of 6.0 m silica (particle diameter 35/45 microns, suspended in 0.1 icoty 5.5 m that corresponds to about 3600 g of silica gel).

Obtaining chiral substrate is carried out as follows, in the column load solution egg serum albumin (1% in 0.15 M phosphate buffer, pH 5.0), followed by absorption in the UV at 280 nm on elyuirovaniya fluid. The quantity of albumin, absorbed on silica gel, as well 200/400 kg Elution performed using 10000 l of 0.15 M phosphate buffer pH 5.0, then 10000 l of 0.15 M phosphate buffer pH 7 and finally, 10000 l of 0.15 M phosphate buffer pH 5.0. Then add 5% solution of the calcium salt of 5-formyl-(6R,S)-tetrahydrofolate acid, equivalent to 5 kg (expiry time is 200/400 l/h), elwira it together with 0.15 M phosphate buffer with pH 5.0. Collect fractions of 500 HP

(6S)-fractions elute earlier (6R)-fractions. Elution fractions can go to control by means of the polarimeter equipped with a flow cell, and then accurately dosed using liquid chromatography under high pressure (HPLC). Enough pure fractions allocate for the required purposes. Pure fractions are collected and transferred to salt by known methods.

Output: 1,9 (28%) (6S)-folinata calcium, Title: HPLC>98%. Optical purity >97%.

Example 2. In this example, play the stationary phase, the method is carried out as in example 1. Elution is carried out, using 10000 l of 0.15 M phosphate buffer with a pH of 7. In the column add 5% solution of the calcium salt of (6R,S)-folic acid, equivalent to about 5 kg at a speed of expiration 200/400 l/h Elution spend phosphate buffer with pH 7. Collect fractions of 500 HP

Separation is unsatisfactory; the cleaning cycle is repeated four times, trying to get a partial separation. Text drop due to insufficient separation.

Example 3. Used the same as in example 1 type columns and the same method of preparation of the stationary phase, but is used in this example, porcine serum albumin (PSA).

Output: 1.5 kg (30%) (6S)-folinata calcium. HPLC titre: >98%. Optical purity: >97%.

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

For elution of use 10000 l of 0.15 M phosphate buffer pH 5.2, with it add 5% solution of (6S)-methyl-tetrahydrofolate acid, equivalent to 10 kg at a speed of expiration 200/400 l/h; again spend elution of 0.05 M phosphate buffer with a pH of 5.2, collecting fractions lei, collected and processed by known methods.

Output: 3,9 kg (39%) calcium salt of (6S)-methyltetrahydrofolate acid. HPLC titre: >97%. Optical purity: >97%.

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

< / BR>
where X represents CH3or CHO;

R1and R2are H, NH+4alkaline and alkaline earth metals, and R1and R2can be the same or different from each other,

by separating a mixture of (6R, S) diastereoisomer General formula I, including passing through a column of an aqueous solution of albumin, washing the column with an aqueous solution of phosphate buffer, loading a mixture of (6R, S) diastereoisomer formula I and elution (6S) isomer aqueous solution of phosphate buffer, characterized in that the concentration of albumin in the solution is 0.1 - 10 wt.%, washing of the column is carried out in three stages - buffer solution with pH 5, then the buffer solution with pH 7 and finally, the buffer solution with pH 5, the concentration of the solution mixture of (6R, S) diastereoisomer of 0.1 - 10 wt.%, pH buffer solution for elution of 4.8 to 5.8.

2. The method according to p. 1, characterized in that these buffer solutions are phosphation download silica gel with granule size 25 - 60 microns.

4. The method according to p. 3, characterized in that the granule size of the silica gel is 35 to 45 microns.

5. The method according to PP. 1 to 4, characterized in that the concentration of the specified albumin in the aqueous solution is about 1 wt.%, and the concentration of this mixture of diastereoisomers is about 5 wt.%.

6. The method according to PP.1 to 5, characterized in that the albumin selected from the group including porcine, bovine, sheep, rabbit, chicken and egg albumin.

7. The method according to PP.1 - 6, characterized in that the aqueous solution of albumin will bufferinput at pH 5.

8. The method according to PP.1 - 6, characterized in that the aqueous solution of this mixture of diastereoisomers will bufferinput at pH 5.3

 

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