FI67082C - FOERFARANDE FOER FRAMSTAELLNING AV D, 1-5-METHYLETETRAHYDROFOLSYRA OCH SALTER DAERAV - Google Patents

Description

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Patent- och registerstyralsan '* AM5kMu € k ^ <xtiuculiri ^^ Mnnd 28.09.8 ** (32) (33) (31) fyrd-tty weHww-a ^ Ird srlerlw 22.02.77 English-England (GB) 7290/77 (71) Bioresearch Sas del Dr.Livio Camozzi S C., Via Marcona, 37 "Milan,

Italia-ltalien (IT) (72) Federico Gennari, Truccazzone, I ta 1 ia-1 ta 1 ien (IT) (7 ^) Oy Kolster Ab (5 * 0 Method for the preparation of d, 1-5-methyltetrahydrofolic acid and its salts - For the first time 1 to 1-, 1-5-methyltetrahydrofolsyrate and safflower

The present invention relates to an improved process for the preparation of d, 1-N, N- (2-amino-3,4,5,6,7,8-hexahydro-5-methyl-4-oxo-6-pteridinyl) methyl / benzoylglutamic acid and its for the preparation of salts of the general formula coox NH - / 'M — CO - NH - CH (i) ί ι \ = s'

A M CH2 Cl10

HN / \ / I 2 j '? H2

H2NAnAnA2 COOX

B

i li h wherein X is hydrogen, alkali metal or alkaline earth metal equivalent.

From now on, acid (I) is only referred to as 5-methyltetrahydrofolic acid (MTHF).

2 67082

The new process can be used industrially and yields the product simply, economically and in high yields, d, 1-5-methyltetrahydrofolic acid is usually prepared from folic acid by a process comprising the following essential steps: a) folic acid tetrahydrofolic acid b) separation and purification of tetrahydrofolic acid c) tetrahydrofolic acid 5-methyltetrahydrofolic acid d) Separation and purification of 5-methyltetrahydrofolic acid

Step (a) is performed by both catalytic hydrogenation and chemical reduction, especially using alkali borohydrides. (Y. Hetefi et al., Biochem, Prep. 7, 89 (1960); K. G. Serimgeour et al., Biochemistry 5, 1438 (1966)). Regardless of the method used, a mixture of folic acid, tetrahydrofolic acid and dihydrofolic acid containing mainly the former two is generally obtained in these steps and is generally difficult to break down into its components. The only suitable method for performing step (b) uses large chromatographic columns and a series of eluents (L. Jaenicke et al., Z. Physiol, Chem. 326, 168 (1961); JCKeresztesy et al., Biophysic Res. Commun. 5, 286 ( 1961); H. Rudiger et al., FE / BS / Letters 4, 316 (1969)). From the solution to be chromatographed, the excess borohydride is first decomposed by adding an acid, preferably hydrochloric acid or acetic acid.

Methylation of tetrahydrofolic acid (step c) can also be carried out by various methods, the most common and suitable of which involves reaction with formaldehyde followed by reduction with a chemical reducing agent, preferably again with alkali borohydride (W. Sakami et al., Biochem. Prep. 10, 103 (1963)).

The separation and final purification of 5-methyltetrahydrofolic acid (step d) have hitherto always been carried out chromatographically using more or less complicated methods, depending on the nature of the mixture to be purified.

In a variation of this method (JABlair et al., Analytical Biochemistry 34, 376-381 (1970)), step (b) is omitted and the reaction mixture from which the excess borohydride is decomposed is treated directly with formaldehyde and another alkali borohydride. Such a modification does not appear to substantially improve the process, since by omitting step (b) in step (a), the omission of step (b) only results in the formation of a more complex and difficult to separate final mixture, which complicates purification step (d) and about 60% product loss at this stage. . In addition, the product obtained is not as high in purity as is required for pharmaceutical purposes.

Thus, this method, which is currently the best known, has never been applied to non-laboratory scale production where small amounts of substances have been produced for experimental use.

It has now surprisingly been found, and it is an object of the present invention, that it is possible to convert folic acid (a product readily available commercially at an affordable price) to d, 1-5-methyltetrahydrofolic acid in very high yields and pharmaceutically pure without using expensive and complex chromatographic purification by reducing the folic acid in a critical reaction. under conditions which allow the reaction to produce virtually 100% selective tetrahydrofolic acid.

The invention is characterized in that a) folic acid is reduced to tetrahydrofolic acid

NaBH 4 in an alkaline aqueous solution in an inert atmosphere 4 using a weight ratio of NaBH 4: folic acid 0.5: 1-3: 1, reaction temperature 60-80 ° C, pH 7-8 and reaction time 0.5-2 hours, b) tetrahydrofolic acid is methylated 5-methyltetrahydrofolic acid with an aqueous solution of formaldehyde and sodium borohydride in an inert atmosphere using a weight ratio of total formaldehyde: tetrahydrofolic acid is 0.8: 1-2.5: 1, weight ratio NaBH 4: tetrahydrofolic acid 0.25: 1-1.5: 1, temperature 25 - 30 ° C, pH 6-8, with formaldehyde being added in successive fractions, of which the fraction with a weight ratio of formaldehyde: tetrahydrofolic acid of 0.15: 1 is added at the end of the reaction and at the end of the step an antioxidant which is cysteine is added. , reduced glutathione or pantethylene, c) sodium 5-methyltetrahydrofolate is isolated from an aqueous solution containing it in the presence of an antioxidant which is cysteine, reduced glutathione or pa ntetein, and that the steps are performed sequentially in the same reactor without isolating the intermediate.

This step can be carried out by two equally advantageous alternative methods, the suitability of each of which is due to the fact that the solution to be treated obtained in step (2) contains practically only sodium 5-methyltetrahydrofolate.

A. Separation by adsorption on activated carbon.

To carry out this step, the activated carbon in an amount of 5 to 20 times the weight of the initially treated folic acid is suspended in water and added with stirring to the final reaction mixture in the pH range of 6.5 to 6.8.

The filtered carbon is suspended in a eluent mixture of C 1-4 alcohol or 2-methoxyethanol and water in a ratio of 50: 50-95-5. The eluent mixture also contains a small amount (0.2 to 5%) of a reducing agent and a small amount (0.5 to 5%) of ammonia in aqueous solution. Sodium 5-methyltetrahydrofolate is recovered from the effluent mixture obtained from carbon by precipitation as a water-miscible organic solvent or by concentrating and lyophilizing the solution or also as an alkaline earth salt from which 5-methyltetrahydrofolic acid can be liberated.

In option A, the preferred conditions for separation and purification of the product are as follows: - ratio of activated carbon: folic acid 7: 1 - eluent mixture containing ethanol, methanol or 2-methoxyethanol and water in a ratio of 80:20, v / v - the reducing agent in the reaction mixture is cysteine up to 1% of the total eluent mixture - the amount of 34% aqueous ammonia mixture is up to 2% of the total eluent mixture (0.7% ammonia).

5 67082 B. Separation by precipitation of 5-methyltetrahydrofolic acid directly from the solution containing it as an alkaline earth metal salt, in particular as a calcium salt.

This alternative, which is even simpler than option A, is particularly suitable when steps (1) and (2) are performed in an aqueous medium at a high concentration and the resulting 5-methyltetrahydrofolic acid must be separated from the concentrated aqueous solution.

The solution from step (2) is treated with a concentrated aqueous solution of CaCl 2 and the solution containing a small amount of reducing agent is left for about 12 hours at 0-5 ° C. The precipitated calcium mesyl tetrahydrofolate pentahydrate is purified by dissolving in a small amount of boiling water and allowing to crystallize at 0-5 ° C. The preferred ratio of folic acid: CaCl 2 is 2: 1 to 3: 1, preferably 2.5. The reducing agent is preferably cysteine, cystamine, reduced glutamine, panthein.

The purity of the product obtained by the method described above is 99%, and the yields are always more than 80%, based on the folic acid in the reaction.

The steps described above are performed sequentially in the same reactor without isolating the intermediates. Reported ratios are always weight ratios.

Comparing the new method to the best known method, the essential steps of which were summarized at the beginning of the description, it can be immediately seen that the chromatographic separation and purification, both intermediate and final, can be omitted in the new method. This not only simplifies and makes the method more economical, as the known method requires long and expensive columns and long process times, but also significantly improves the yields due to the high product losses in the chromatographic separation.

Additional losses occur when the product is recovered from very dilute solutions. Recovery is then also apparently quite expensive. The final separation and purification step has been replaced by an adsorption / desorption step with activated carbon or precipitation with Ca salts, which are simple methods with good yields. These steps are completely new in the preparation of 5-methyltetrahydrofolic acid and its salts.

From the above description, it can be seen that the whole production process of 5-methyl-tetrahydrofolic acid is carried out in the presence of an inert gas and in which steps in the presence of a reducing agent.

This is due to the need to prevent the self-oxidation of tetrahydrofolic acid or 5-methyl-tetrahydrofolic acid to the corresponding dihydrofolic or folic acids. The same problem of self-oxidation applies to the storage of 5-methyl-tetrahydrofolic acid.

To date, the reducing agents proposed for the storage of MTHF include 2-mercaptoethanol and ascorbic acid. The use of these substances apparently led to the conclusion that the product was not intended for pharmaceutical use but was limited to laboratory use. Namely, 2-mercaptoethanol is toxic and has a disgusting odor, while ascorbic acid decomposes rapidly at the optimum pH of injection solutions (7-8).

Another object of this invention is a pharmaceutical composition of a stabilized alkali or alkaline earth metal 5-methyltetrahydrofolate.

The novel stable compositions contain essentially 5-methyl ethyl tetrahydrofolate (as active ingredient) and 5-50% of an organic compound of biological origin (as stabilizer) containing at least one -SH group. Typical of such compounds are cytheine, cystamine, panthein, reduced glutathione, and all derivatives thereof in which the -SH function has remained active.

All of the above substances are non-toxic, stable at physiological pH ranges and thus suitable for use in liquid or solid pharmaceutical preparations for human use.

The novel pharmaceutical compositions have an effect on red blood cell formation, a liver protective effect and an antineoplastic effect.

The following are some examples of the implementation of the method according to the invention in order to better illustrate the method. *

Example 1 30 liters of deionized water are fed to a reactor, and 2.4 kg of d, 1-folic acid are suspended in water. Solid Ha 2 CO 2 O 3 is then added with stirring until the folic acid is completely dissolved (pH 7.3 of the final solution), then 4.3 kg of NaBH 4 dissolved in 7,670,82 in 40 liters of water are added and the reaction mixture is heated at 70 ° C. The mixture is allowed to react for one hour with stirring and continuous conduction of nitrogen. After completion of the reaction, the reactor is cooled to 30 [deg.] C. and 3.5 kg of 37% aqueous formaldehyde and 2.4 kg of sodium bolohydride dissolved in 20 liters of water are added with stirring. The reaction is allowed to proceed for one hour with stirring and introducing nitrogen into the reaction mixture. After this time, an additional 0.15 kg of 37% formaldehyde and 1 kg of cysteine are added.

17 kg of activated carbon are suspended in 100 liters of air-free water, and the carbon suspension is added with stirring to the reaction mixture, the pH of which has been adjusted to 6.5. After a few minutes, the mixture is filtered under nitrogen and the filter cake is washed with water containing 1% cysteine until all inorganic salts have been completely removed. The carbon is suspended in 100 liters of an eluent mixture having the following composition by volume: 2-methoxyethanol 80, water (containing 5% cysteine) 20, ammonia (34%) 2.

The suspension is stirred for a few minutes, then filtered and the filtrate is concentrated to 20 liters. The concentrated filtrate is poured into a solution containing 700 g of CaCl 2 per 100 liters of ethanol.

Calcium 5-methyltetrahydrofolate precipitates and is filtered under nitrogen, washed with ethanol and dried in vacuo. 2.3 kg of product are obtained, which corresponds to a yield of 86% with respect to the starting folic acid.

* 5

UV analysis of the product: (pH - 7; £ _ = 32.10): maximum absorption: 290 nm minimum absorption: 245 nm ratio E29q / E245 = 3'8 *

Chromatographic analysis by the method of Nixon and Bertino (P. F. Nixon, J. R. Bertino, Methods in Enzym. 18, 661 (1971)) using a Sephadex DEAE A-25 chromatography column gave only a 5-methyltetrahydrofolic acid peak.

High performance liquid chromatography (HPLC analysis) (Partisil-10-SAX 4.6 x 250 mm column; eluent 5% ammonium citrate; pH 6) gave only a 5-methyltetrahydrofolic acid peak.

NMR Spectrum: Typical singlet of the N 2 -CH 2 group: δ 51 7.5.

1 kg of the calcium 5-methyltetrahydrofolate thus prepared is dissolved by heating under nitrogen in 40 liters of water containing 1 kg of cysteine. The pH of the solution is adjusted to 6 with dilute hydrochloric acid, 8 67082 and the mixture is allowed to stand in the refrigerator. The precipitated 5-methyltetrahydrofolic acid is filtered off, washed with a small amount of cold water and dried. The product ratio E290 ^ E245 is

The same procedure is repeated using ethanol, methanol, 1- or 2-propanol, n-butanol, tert-butanol, sec-butanol or isobutanol instead of 2-methoxyethanol in the eluent mixture. In all cases, a product with the same properties was obtained in equally good yield.

Example 2 5-Methyltetrahydrofolate is prepared as described in Example 1. Carbon is eluted with a mixture of 80 volumes of methanol, 20 volumes of water (with 5% reduced glutathione) and 2 volumes of ammonia (34%).

After filtration of the activated carbon, 700 g of CaCl2 are added to the eluate, relying on a small amount of water.

Calcium 5-methyltetrahydrofolate precipitates and is filtered under nitrogen, washed with ethanol and dried in vacuo. 2.1 kg of product are obtained, which corresponds to a yield of 80% with respect to folic acid. The product has the same typical properties as the product obtained in Example 1.

Example 3

The procedure of Example 1 is repeated until the treatment of the carbon cake is carried out with a mixture of eluent containing 2-propanol and water with a small amount of ammonium hydroxide and cysteine, the eluate is filtered and then concentrated to 20 liters.

This solution is lyophilized to give 2.1 kg of sodium 5-methyltetrahydrofolate, yield 80%.

The product has E29o / E245 = 3'® *

Example 4

The procedure of Example 1 is repeated until the eluate is concentrated. In this experiment, the eluate is treated with 1.3 kg of BaCl2 dissolved in 100 liters of ethanol. Barium-5-methyltetrahydrofolate precipitates, yield 83%. E290 ^ 245 = 3 '^ 3,

Example 5

The procedure of Example 1 is repeated until the eluate is concentrated. In this experiment, the eluate is treated with 0.6 kg of MgCl2 dissolved in 100 liters of ethanol. Magnesium 5-methyltetrahydrofolate precipitates, yield 82%. Ε290 ^ Ε245 = 3'® * 9 67082

Example 6 100 g of calcium 5-methyl tetrahydrofolate prepared according to Example 1 are dissolved in 4 liters of water. A stoichiometric amount of Na 2 SO 4 is added to the solution. CaSO 4 precipitates and is filtered.

The clear solution is lyophilized to give sodium 5-methyl tetrahydrofolate.

Example 7 Preparation of the calcium salt of MTHF acid 2.50 kg of folic acid are suspended in 10 liters of water, 1 liter of a 40% Na 2 CO 3 solution is added to the suspension with stirring. The pH is adjusted to 7.8 with Na2CO3, the solution is cooled and 1.70 kg of NaBH4 are added under a nitrogen atmosphere. The mixture is allowed to react at about 70 ° C for 2 hours, at which time the reaction is complete. 4.00 kg of 37% aqueous formaldehyde are then added, followed by 0.80 kg of sodium borohydride. The precipitated sodium borate is filtered off and the excess borohydride is discarded by adjusting the pH of the mixture to 6 with HCl. To the resulting reaction mixture is added with stirring 1 kg of CaCl 2 dissolved in 2 liters of water and the mixture is allowed to stand at 3 ° C overnight.

Calcium methyl tetrahydrofolate precipitates as an amorphous white powder, is filtered and washed with water. This salt is dissolved in 10 liters of boiling water, the solution is cooled and left to stand overnight. The precipitated crystalline salt is filtered off, washed and dried in vacuo. 2.40 kg of calcium methyl tetrahydrofolate pentahydrate are obtained in a total yield of 80%, based on the folic acid used as starting material.

Under the same conditions, the Mg salt can be prepared, which, however, is difficult to filter.

As mentioned above, the 5-methyltetrahydrofolate and its salts prepared by the process of the invention can be prepared into stable therapeutic compositions at room temperature by mixing 5-50% of a stabilizer which is a compound of biological origin having at least one -SH group.

Typical such compounds are cysteine, cystamine, panthein and reduced glutathione.

To illustrate this aspect of the invention, a typical composition that can be prepared using these compounds is set forth below.

10 67082

Example 8

Pharmaceutical preparation for oral use Tablet

Active dose 25 mg

Stabilizer 12.5 mg

Excipients for the manufacture of the tablet 112.5 mg Pharmaceutical preparation for injection

Active substance 25 mg / ml

Stabilizer 12.5 mg / ml

Lyophilization vehicle 150 mg / ml

If necessary, the pH of the solution is adjusted to 7.5 with concentrated NaOH, the solution is filled into vials and lyophilized.

The term active substance means - 5-methyltetrahydrofolic acid - sodium 5-methyltetrahydrofolic acid - calcium-5-methyltetrahydrofolic acid - magnesium-5-methyltetrahydrofolic acid

The term "stabilizer" in this example means - cysteine - cystamine - panthein - reduced glutathione.

The term "lyophilization carrier" means any substance of this definition commonly used in a lyophilization technique, such as glycine.

In no case was no change in the active substance observed in the solution during storage in the vial or during lyophilization.

Claims (9)

A process for the preparation of 1,1,5-methyltetra-hydrofolic acid and its salts of the general formula (I) COOX n Pu NH - / CO-NH-CH? wherein X is hydrogen, an alkali metal or an equivalent of an alkaline earth metal, characterized in that a) folic acid is reduced to tetrahydrofolic acid by f03 l / _ / IA CH0 HN NaBH4 in an alkaline aqueous solution in an inert gas atmosphere using a weight ratio NaBH ^: folic acid of 0.5: 1 - 3: 1, a reaction temperature of 60-80 ° C, a pH of 7-8 and a reaction time of 0.5 - 2 hours, b) the tetrahydrofolic acid is methylated to 5-methyltetrahydrofolic acid with an aqueous solution of formaldehyde and sodium borohydride in an inert gas atmosphere using a weight ratio total amount of formaldehyde: tetrahydrofolic acid of 0.25: 1-1.5: 1, a temperature of 25-30 ° C, a pH of 6-8, wherein the formaldehyde is added in the form of successive fractions, the fraction having a weight ratio formaldehyde: tetrahydrofolic acid of 0.15: 1 being added after the reaction is completed; and at the end of the step, an antioxidant is added, c) the sodium 5-methyltetrahydrofolate is isolated from the aqueous solution containing the same in the presence of an antioxidant, which is cysteine, reduced glutathione or pantetein, reactor without isolating the intermediate. 2. Process according to claim 1, characterized in that the separation step c) is carried out by treating the reaction mixture with an amount of activated carbon with a weight of 5-20 times the weight of the initially treated folic acid, and The 5-methyltetrahydrofolate is desorbed from the carbon with an eluent containing Cj C alcohol or 2-methoxyethanol and water in a weight ratio of 50: 50 - 95: 5. 3. A process according to claim 1, characterized in that the separation step c) is carried out by treating the aqueous solution with a Ca salt and cooling the solution to 0-5 ° C, the weight ratio of folic acid: calcium salt being 2: 1-3: 1st 4. Process according to claim 1, characterized in that in step a) the weight ratio NaBHBH: folic acid is 0.7: 1-2: 1, the temperature is 70 ° C, pH 7-8 and the reaction time is 1 hour and that in step b) the weight ratio is the total amount of 37 -%: in formaldehyde: tetrahydrofolic acid 1.4: 1, the weight ratio NaBH 5. A process according to claim 2, characterized in that in step c), the amount of activated carbon 7 is multiplied by the weight of the initially treated folic acid and, as eluent, a mixture containing methanol, ethanol, 2-methoxyethanol, 1- or 2-propanol, n-butanol, tert-butanol, sec-butanol or isobutanol and water at a ratio of 80: 20 w / v and containing 1% by volume of an antioxidant and 0.7% ammonia in an aqueous solution. 6. A process according to claim 1, characterized in that 5-methyltetrahydrofolic acid is precipitated from an aqueous solution in the form of an alkali metal or alkaline earth metal by acidifying the aqueous solution. 7. A process according to claim 2, characterized in that the 5-methyltetrahydrofolic acid is obtained in the form of sodium salt by concentrating the eluate and lyophilizing the concentrate.