CA1093554A

CA1093554A - Process for producing d,1-5-methyltetrahy-drofolic acid and its salts

Info

Publication number: CA1093554A
Application number: CA296,754A
Authority: CA
Inventors: Federico Gennari
Original assignee: Bioresearch Sas Del Dr Livio Camozzi & C
Current assignee: Bioresearch Sas Del Dr Livio Camozzi & C
Priority date: 1977-02-22
Filing date: 1978-02-09
Publication date: 1981-01-13
Also published as: FR2381047A1; DK76578A; FR2385397A1; PL118654B1; FR2385397B1; SE7801973L; ZA78837B; NL190285C; AT380020B; AU3343078A; FR2381047B1; ATA90378A; AR215162A1; FI67082C; AU520715B2; NO780594L; YU40505B; CZ112978A3; NL190285B; BE863808A

Abstract

ABSTRACT
A process for producing d,1-5-methyltetrahydrofolic acid and its salts of the formula (I)

Description

355~

This invention relates to an improvedprocess for producing d, l-N- [p- (2-amino-3,4,5,6,7,8-hexahydro-5-methyl-4-oxo-6-pteridinyl)methyl~benzoyl glutamic acid and its salts of formula COQX
NH ~ CO-NH - CH
5:~ I H3 ¦ \=/ 1H ( I) HNJ~ ~H2 t 2 H2N~ , COOX

H

where X is hydrogen, an alkaline metal or the equivalent of an alkaline earth metal.
Hereinafter, the acid (I) will be referred to only as 5-methyl-tetrahydrofolic acid (MTHF).
The new process is applicable industrially for ob-taining a high purity product simply, economically and with high yields. Notably, d,l-5-methyltetrahydrofolic acid is commonly prepared from folic acid by a process comprising the following essential stages:
i id reductiOn > tetrahydrofOlic acid b) separation and purification of the tetrahydrofolic acid c) tetrahydrofolic acid methylation 3 5-methyltetrahydro-folic acid d) separation and purification of the 5-methyltetrahydrofolic ~`

acid.
The stage (a) has been carried out both by catalytic hydrogenation and by chemical reduction, in particular by using alkaline borohydrides. (Y. Hetefi et al. - Biochem. Prep. 7, 89 (1960); K.G. Serimgeour et al. - Biochemistry 5, 1438 (1966~). In this stage, no makter what known method is usedt a mixture of folic acid, tekrahydrofolic acid ---~---- -------,~

5~

and dihydrofolic acid is obtained, consisting mainly of -the first two components and difficult to resolve. The only useful methods comprise carrying out stage (b) with very large chromatography columns and using a series of eluen,s (L. Jaenicke et al. - Z.physiol. Chem. 326, 168 (1961)~ J.C. Keresztesy et al. - Biochem. Biophysic Res. Commun. 5, 286 (1961);
H. Rudiger et al. FE/sS/Letters 4, 316 (19~9)~ The excess of borohydride in the solution subjected to chromatographic separation is firstly destroyed by an acid, preferably hydro-chloric acid or acetic acid. `
The methylation of the tetrahydrofolic acid (stage c)may also be carried out by various methods, the most common and convenient of which comprises reaction with formaldehyde and then reduction with a chemical reducing agent, preferably again an alkaline borohydride (W. Sakami et al., Biochem. Prep.
10, 103 (1963)).
The separation and final purification of the 5-methyltetrahydrofolic acid (stage d) has always been carried out up to the present time by chromatography processes of a greater or lesser complexity, as determined by the nature of !
the mixture to be purified.
In one modification of said method (J.A. slair et al., Analytical Biochemistry 34, 376~381 (1970)), stage (b) is dispensed with and the reaction mixture in which the excess borohydride has been destroyed is treated directly with formaldehyde and another alkaline borohydride. Evidently such a modification does not substantially improve the process, because as stage (a) in any case leads to the production of a mixture as heretofore specified, dispensing with stage (b) gives a final mixture which is even more complex and difficult to resolve, with consequent aggravation of the purification stage (d), and a loss in this stage of about 60% of the product.
Furthermore, a high purify product is not obtained as would be required for use in the pharmaceutical field.

~355i9~
In conclusion, said process, which is the best known at the present time, has never gone beyond laboratory use and the production of small quantities of substance for experimental use.
It has now been surprisingly found and forms the object of the present invention, that it is possible to transform folic acid (a product easily obtainable commercially at economically convenient prices) into d,l-5-methyltetra-hydrofolic acid with very high yields and of pharmaceutical purity and without the use of costly and complicated s-tages of chromatographic purification, if the folic acid is reduced under critical reaction conditions which make it practically 100% selective in producing tetrahydrofolic acid.
~ The process according to the present invention comprises essentially the following stages:
1. Reduction of folic acid to tetrahydrofolic acid by sodium borohydride in an aqueous alkaline solution and an inert gas atmosphere, at a temperature between 60 and 80C, The ratio of NaBH4: folic acid lies between 0.5:1 and 3:1, and the reaction time between 30 minutes and 2 hours.
On this basis, the preferred reaction conditions are as follows: ;~
- ratio of NaBH4: folic acid 0.7:1, 2:1 - reaction time 1 hour - pH 7-8 - temperature 70C.

2. Methylation of the tetrahydrofolic acid to 5-methyl-tetrahydrofolic acid by treatment with a 37~ aqueous solution of formaldehyde and then again with sodium borohydride.
At the end of the reaction 37% formaldehyde is again added.
In this stage the cri~ical reaction conditions are as follows:

s~
- pH 6 to 8 - tempera-ture 25-30C
- ratio of 37~ formaldehyde: tetrahydrofolic acid between 0.8:1 and 2.5:1 - ratio of NaBH4:tetrahydrofolic acid between 0.25:1-1.5:1 - inert gas atmosphere - presence of a reducing agent in the final reaction stage (cysteine, reduced glutathione, pantetheine).
On the above basis, the preferred reaction conditions are as follows:
- pH 6.5 - temperature 30C
- ratio of 37~ formaldehyde: tetrahydrofolic acid 1.4:1 .
- ratio of NasH4: tetrahydrofolic acid included between 0.5:1 and 1:1 - reducing agent present in the preaction mixture: cysteine.

3. Separation and purification of the sodium 5-methyl-tetrahydrofolate. This step may be performed following two equally advantageous alternatives both allowed by the fact that the solution to be treated, obtained in step (2), does practically contain only sodium 5-methyltetrahydrofolate:
A-Separation by adsorption/desorption on active carbon.
To carry out this stage, a quantity of active carbon equal to 5-20 times the weight of the initially treated folic acid is made into a pulp in water and added to the final reaction mixture at pH 6.5-6.8 under agitation.
The filtered carbon is suspended in a~ eluent mixture consisting of an alcohol (containing 1 to 4 carbon atoms) or 2 methoxy-ethanol and water in a ratio between 50/50 and 95/5. The eluent mixture also contains a small percentage (0.2 to 1.5%) of a reducing agent, and a small percentage (0.5 to 5%) of ammonia in aqueous solution. The sodium 5-methyltetrahydrofolate may be recovered from the effluent mixture filtered from the carbon by precipation with an ~93559~
organic solvent miscible with water, or by concentrating the solution and lyophilising, or again in the form of an alkaline earth salt, from which the 5-methyltetrahydrofolic acid may be release~l.
On the above basis the preferred conditions for the sepa-ration and purification stage according to this alternative A are as follows:
- ratio of active carbon: folic acid 7:1 - eluent mixture consisting of ethanol, methanol or 2-methoxy-ethanol and water in a ratio of 80/20, v/v - reducing agent in the ~eaction mixture: cysteine to the extent of 1% of the total eluent mixture - 34% aqueous ammonia solution to the extent of 2% of the total eluent mixture (0.7% of ammonia).
B ~ Separation by direct precipitation of the 5-methyltetrahydro-folic acid from the solution containing it, as alXaline-earth metal salts, in particular as calcium salt~
This alternative, still more simple than the alternative A, is particularly useful when the steps (1) and (2) have been carried out in a concentrated aqueous medium and thus the 5-methyltetra-hydrofolic acid has to be separated from a concentrated aqueous `~
solution. `~
The solution resulting from stage (2) is treated with a concentra-ted aqueous solution of CaCl and left to stay over about 12 hours at a temperature between 0 and 5C in the presence of a little amount of a reducing product.
The thus precipitated calcium methyl-tetrahydrofolate pentahydrate is purified by dissolution in a little volume of boiling water and recrystallized by leaving the solution at 0-5C.

3 The preferred ratio folic acid: caCl i5 comprised between 2:1 ~ 5 --355~
and 3:1 and is preferably of 2.5.
The reducing product is preferably selected in the group consisting of cysteine, cysteamine7 reduced glutathione, pantetheine.
The product obtained by the new process described has a purity exceeding 9y% and is obtained with yields always exceeding 80~ with respect to the initial folic acid.
The stages heretofore described are carried out in suc-cesslon in the same reactor, without separating~intelmediate products.
The ratios indicated are always ratios by weight.
Comparing the new process according to the invention with the best process of the known art, the essential stages of which were summarised at the beginning of this specification, it is immediately apparent that the new process enables both the intermediate stage and final stage of c:hromatographic separation and purification to be dispersed with.
This not only leads to c~nsiclerable simplification and economy of the process, the known process requiring long and costly columns and long processing times J but also gives a considerable increase in yields because the chromatographic separation gives large losses of useful product.
Further losses arise in recovering the product from its very dilute solutions. Such recovery is also obviously very costly.
The final separation and purification stage is replaced by the adsorption/desorption stage on active carbon or by the precipitation with Ca salts which are extremely simple and of high yield. These stages are completely new in the production of 5-methyltetrahydrofolic acid and its salts~
39 From the description given heretoforeJ it can be seen 3~iS~
that the entire production process for 5-methyltetrahydrofolic acid is conducted in the presence of inert gas, and in some stages in the presence of a reducing agent. This is due to the need to prevent self-oxidation either of the tetrahydrofolic acid or of the 5-methyltetrahydrofolic acid to the corresponding dihydrofolic and folic acids.
The same self-oxidation problem occurs in preserving the 5-methyltetrahydrofolic acid.
Up to the present time, the reducing age~ts suggested for conserving MTHF acid were 2-mercaptoethanol and ascorbic acid.
obviously these substances meant that no industrial use of the product in the pharmaceutical field could be considered, and were limited to solving a laboratory problem.
In this respect. 2-mercaptoethanol is a toxic product of nauseating odour, while ascorbic acid decomposes rapidly at the optimum pH values (7-8) for injectable solutions.
A further object of the present invention is to provide stabilised compositions of alkaline or alkaline earth 5-methyl-tetrahydrofolates ~or pharmaceutical use The new stable compositions consist essentially of `
5-methyltetrahydrofolate (as active substance) and 5 to 50~ of an organic compound of biological origincontaining at least 1 ~SH
group (as stabiliser). Characteristic compounds of this class are for example cysteine~ cysteamine, pantetheine~ reduced gluta-thi~ne and all their derivatives in the -SH function is maintained active.
All the aforesaid substances are without toxicity, are stable at physiological pH values and are thereforesuitable for the preparation of liquid or solid pharmaceutical compounds for any type of application in the human field.

9355~
The new pharmaceutical compositions possess hemopoietic action, protective activity on the liver and antineoplastic activity.
Some practical examples of the process are given hereinafter in order to better illustrate and facilitate the reproduction of the process according to the present invention.
It is apparent that these examples are not limiting~
and the reaction conditions may be varied within the limits de-fined in the specification and incorporate all the expedients well known or immediately evident to an expert of the art.

80 1 of deionised water are fed into a reactor and 2~4 kg. of d~l-folic acid are suspended therein. Solid Na2C03 is added under agitation until the folic acid is completely dissolved (final solution . . . . . . . . . . . . . . . . . . . . . . . . .

7a -~3~5~

pH 7.8), then 4.8 kg of NaBH4 dissolved in 40 1 of H2O are added and the reaction mixture heated to 70C. The mixture is reacted for 1 hour under agitation while constantly under a stream of nitrogen. When the reaction is finished the re-actor is cooled to 30C and 3.5 kg of 37% aqueous formalde-hyde and 2.4 kg of sodium borohydride dissolved in 20 1 of water are added under agitation. The reaction is continued for 1 hour under agitation in a stream of nitrogen. After this time, a further 0.35 kg of 37% formaldehyde and 1 kg of cysteine are added.
17 kg of active carbon are suspended in 100 1 of de-aerated water and the carbon suspension is added under agita-tion to the reaction mixture, brought to pH 6.5. ~fter a few minutes it is filtered under nitrogen and the carbon cake on the filter is washed with water containing 1% of cysteine until all the inorganic salts have been completely removed. The carbon is suspended in 100 1 of an e:Luent mixture of the fol-lowing volumetric composition:
2-methoxy-ethanol 80, water (contain:ing 5% of cysteine) 20, ammonia (34%) 2r The suspension is left under agitation for some minutes, is then filtered and the filtrate concentrated to a volume of 20 litres. The concentrated filtrate is poured into a solution containing 700 g of CaC12 in 100 litres of ethanol.
Calcium 5-methyltetrahydrofolate precipitates and is filtered off under nitrogen, washed with ethanol and then dried under vacuum. 2.3 kg of product are obtained, equal to a yield of 86% on the initial folic acid.
On U.V. analysis the product shows the following 30 characteristics (pH -- 7; ~ = 32.10 ):
maximum absorption at 290 nm minimum absorption at 245 nm rati E290/E245 , . . .

Analysis over a Sephadex* ~EAE A-25 chromatographic column using the method of Nixon and Bertino (P.F. Nixon, J.R.
Bertiro, Methods in Enzym. 18, 661 (1971)) shows only the 5-methyltetrahydrofolic acid peak. HPLC analysis (Partisil-lOSAX

4.6 x 250 mm column; eluent 5% ammonium citrate; pH = 6) shows only the 5~methyltetrahydrofolic acid peak. NMR spec-trum: singlet characteristic of the N5-CH3 group at I = 7.5.
1 kg of the calcium 5-methyltetrahydrofolate prepared in this manner is dissolved in 40 1 of water containing 1 kg of cys-LO teine under heat and nitrogen.
The pH is brought to 6 with dilu-te hydrochloric acid and the mixture allowed to stand in a refrigerator.

5-methyltetrahydrofolic acid precipitates and is filtered off, washed with a little cold water and dried. The product shows an E290/E245 value of 3-8-The same process was repeated but replacing the 2-methoxy-ethanol in the eluent mixture with ethanol r methanol, 1 or 2-propanol, n butanol, t-butanol, sec-butanol and iso-butanol respectively. In each case a product was obtained of the same characteristics and the same y2eld.

5-methyltetrahydrofolate is prepared as described under Example 1. The carbon is eluted with a mixture of the following composition: methanol 80, water (containing 5% of reduced glutathione) 20, ammonia (34%) 2.
After filtering the active carbon, 700 g of CaC12 dissolved in a little wa-ter are added to the eluate.
Calcium 5-methyltetrahydrofolate precipitates and is filtered off under a stream of nitrogen, washed with ethanol and dried under vacuum.

2.1 kg of product are obtained, equal to a yield of 80% on the folic acid.
* Trade Mark _ g _ " ~ .

~3~;S~
The product has the same characteristics as that obtained in Example 1.

The preparation is repeated as described under Example 1 as far as the treatment of the carbon cake with the eluent mixture consisting of 2-propanol and water containing small percentages of ammonium hydrate and cysteine~ the filtered eluate then being concentrated to a volume of 20 litres.
This solution is subjected to lyophylisation to give 2.1 kg of sodium 5-methyltetrahydrofolate with a yield of 80%.

Example 1 is followed as fax as the concentration of the eluate. In this te9t the eluate is treated with 1.3 kg of BaC12 dissolved in 100 litres of ethanol.
Barium 5-methyltetxahydrofolate precipitates with a yield of ~,3%. E E = 3.79.

EXAMPLE ~
Example 1 is followed as far as the concentration of the eluate. In this test the eluate is treated with 0.6 kg of MgCl dissolved in 100 litres of ethanol.
Magnesium 5-methyltetrahydrofolate precipitates with a yield of 8,2~. E E = 3.8.

100 grams of calcium 5-methyltetrahydrofolate prepared as described under Example 1 are dissolved in 4 litres of H 0.
The stoichiometric quantity of Na S0 is added to this solution.

~aS0~ is precipitated and separated by filtration~
The clear solution is subjectedto lyophilisation~ to give sodium 5-methyltetrahydrofolate.

Preparation of the calcium salt of MTHF acid.
2~50 Xg of the folic acid are suspended into 10 1 of water and 1 1 of 40~ Na2C03 is added under stirring.
The pH is adjusted to 7.~, with Na2C0 and, after cooling, 1.700 kg of NaBH are added under nitrogen atmosphere.
The rnixture is left to react over 2 hours while heating at about 70C to complete the reaction; 4.00 kg of 37~ aqueous ~;
formaldehyde are added and thereafter 0.80 kg of sodium borohydride.
The precipitated sodium borate is filtered and the excess borohydride is destroyed by adjusting the pH of the solution at .

6 with HCl. 1 kg of CaCl dissolved into 2 1 of water are added while stirring and the obtained reaction mixture is left at 3C
overnight. The calcium methyltetrahydxofolate precipitates as an amorphous white powder which is filtered and washed with water.
This salt is dissolved into 10 1 of boiling water and the cooled solution is left to rest overnight.
The precipitated crystalline salt is filtered, washed and dri~d under vacuum.
2.40 kg of calcium methyl~tetrahydrofolate pentahydrate are obtai~ed with an overall yield of 80~ over the starting folic acid.
Under the identical conditions also the Mg salt is obtained which however is difficult to filtrate.

3~
As stated, 5-methyltetrahydrofolate acid or its salts obtained by the present process can form stable therapeutic compositions at ambient temperature if mixed with 5 to 50% of a stabilising substance chosen from the group consisting of compounds of biological origin containing at least 1 -SH group.
Characteristic compounds of this class are cysteine, cysteamine, pantetheine and reduced glutathione.
In order to illustrate this aspect of the invention, a typical formulation which can be prepared using the aforesaid compound is given hereinafter.

Pharmaceutical preparation in oral form Tablets Active substance 25 mg Stabiliser 12.5 mg Excipients for compression, quantity necessary to make up 150 mg.
Pharmaceutical pr-eparation in injectable form Active compound 25 mg/ml Stabiliser 12.5 mg/ml Lyophilisation support 150 mg/ml When necessary~ the solution is brough-t to pH 7.5 with concentrated NaOH, fed into a phial and lyophilised.
The term "active compound" signifies:
- 5-methyltetrahydrofolic acid ~ sodium 5-methyltetrahydrofolate - calcium 5-methyltetrahydrofolate - magnesium 5-methyltetrahydrofolate The term "stabiliserl' in this example signifies:

- cysteine - cysteamine .

ss~
- pantetheine - reduced glutathione The term "lyophilisation support" signifies any substance which ~ay fall within this definition in usual lyophilisation technology, such as glycine.
In no case did the solution show any alteration of the active product during its retention in the phial or during lyophilisation.

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. An improved process for producing d,1-5-methyl-tetrahydrofolic acid and its salts of formula (I) where X is hydrogen, an alkaline metal or the equivalent of an alkaline earth metal, wherein a. folic acid is reduced to tetrahydrofolic acid with NaBH4 in an aqueous alkaline solution and under an inert gas atmosphere, using a NaBH4: folic acid ratio. of 0.5:1 to 3:1, a reaction temperature of 60 to 80°C and a reaction time of 30 minutes to 2 hours;
b. the tetrahydrofolic acid is methylated to 5-methyltetrahydrofolic acid with aqueous solutions of formalde-hyde and sodium borohydride in an inert gas atmosphere, using a total formaldehyde : tetrahydrofolic acid ratio. of 0.8 : 1 to 2.5 : 1, a NaBH4 : tetrahydrofolic acid ratio of 0.25:1 to 1.5:1, a temperature of 25 to 30°C and a pH of 6 to 8, the formaldehyde being added in successive fractions, one of which, of formaldehyde : tetrahydrofolic acid ratio 0.15:1, is added when the reaction is terminated;
c. the sodium 5-methyltetrahydrofolate is separated from the aqueous solution containing it, said stages being all carried out in succession in a single reactor without separa-tion of intermediate products.

2. Process according to claim 1 wherein the step (c) of separation is performed by treating the reaction mixture with a quantity of active carbon corresponding to 5-20 times by weight of the initially treated folic acid, and desorbing the sodium 5-methyltetrahydrofolate from the carbon by an eluent consisting of an alcohol containing 1 to 4 carbon atoms or 2-methoxy ethanol and water in a ratio of 50/50 to 95/5.

3. Process according to claim 1 wherein the step (c) of separation is performed by treating the reaction mixture with an aqueous solution of a Ca salt and cooling the solution at 0°-5°C, the ratio folic acid: calcium salt being included between 2:1 and 3:1.

4. A process as claimed in claim 1, wherein stage (a) is carried out with a NaBH4 : folic acid ratio of 0.7:1, 2:1, a temperature of 70°C, a pH of 7 to 8 and a reaction time of 1 hour and stage (b) is carried out with a total 37% formaldehyde :
tetrahydrofolic acid ratio of 1.4:1, a NaBH4 ; tetrahydrofolic ratio included between 0.5:1 and 1:1, a temperature of 30°C and a pH of 6.5.

5. Process according to claim 2, wherein stage (c) is carried out using a quantity of active carbon equal to 7 times the weight of the initially treated folic acid and an eluent con-sisting of methanol, ethanol, 2-methoxy ethanol, 1- or 2-propanol, n-butanol, t-butanol, sec-butanol or isobutanol and water in a ratio of 80/20, v/v with 1% by volume of a reducing agent and 0.7% of ammonia in aqueous solution.

6. A process as claimed in claim 1, wherein the 5-methyl-tetrahydrofolic acid is precipitated from the aqeuous solution by acidification, in the form of one of its alkaline or alkaline earth salts.

7. A process as claimed in claim 1, wherein the 5-methyl-tetrahydrofolic acid is obtained as its sodium salt by concentrating the eluate and lyophilising the concentrate.

8. A process as claimed in claim 1, wherein the 5-methyl-tetrahydrofolic acid is separated from the eluate by precipitation in the form of an alkaline earth salt.

9. Process according to Claim 3, wherein the puri-fication of calcium 5-methyltetrahydrofolate is carried out by dissolving the salt pentahydrate in little boiling water and cooling the solution at 0° - 5°C.

10. Process according to Claim 3, wherein stage (c) is carried out in the presence of a reducing product.