GB1560372A - Preparation of calcium folinate - Google Patents

Description

(54) PREPARATION OF CALCIUM FOLINATE (71) We, THE WELLCOME FOUNDATION LIMITED, of 183-193 Euston Road, London N.W.1., a company incorporated in England, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is performed, to be particularly described in and by the following statement: Calcium leucovorin is useful in diminishing the toxicity and counteracting the effect of inadvertantly administered overdoses of folic acid antagonists and in the treatment of the megaloblastic anemias due to sprue, nutritional deficiency, pregnancy and infancy (Physicians Desk Reference, 31st Edition 1977, p. 904, 905). Calcium leucovorin is a potent agent for neutralizing the immediate toxic effects of Methotrexate and is therefore useful as a "rescue" agent in reversing the peripheral toxic effects of Methotrexate (or other antifols) resulting from its use in anti-neoplastic chemotherapy.

Calcium leucovorin is an extremely expensive material, mainly because the here tofore available methods of synthesis - yielded a final product which required extensive purification in order to pass USP (United States Pharmacopeia) specifications.

Almost invariably, the prior art purification has required column chromatography which is a very expensive stage in the preparation of calcium leucovorin for a number of reasons. For example, it is time consuming.

labor and equipment intensive, and uses exceedingly large volumes of solvent for elution.

It has now been unexpectedly discovered that if an amine base is used to effect ring opening of anhydroleucovorin to give leucovorin, the calcium leucovorin produced therefrom contains substantially lower levels of impurities than if an inorganic base is used. Surprisingly, in most cases calcium leucovorin prepared using an amine base in the ring opening step was sufficiently pure to pass USP specifications after simply filtering, washing and drying the product, i.e. column chromatography was not required.

An amine base useful in this invention is one which does not give rise to any significant amount of inorganic salts as contaminants of the isolated calcium leucovorin and results in demonstrably less contamination by organic materials as well.

Preferred amine bases include methylamine, triethylamine, morpholine, tetraethylammonium hydroxide, and N,N-diethylethanolamine. Other bases which are suitable but less preferred include ammonia, aniline, p-chloroaniline, n-butylamine, cyclohexylamine, diethanolamine, triethanolamine, diethylamine, dimethylamine, N,N'dimethylaniline, piperidine, piperazine, N-methylpiperazine, N,N'-dimethylpiperazine, pyridine, imidazole, benzylamine, ethylenediamine, o-, m-, and p-toluidine, > , m-, and p-anisidine. In addition it should be understood that those skilled in the art would be able to select other amine bases suitable for use in the process of this invention.

When ammonia is used in the practice of this invention, it may be desirable to operate in pressure equipment to prevent loss of ammonia by evaporation. Indeed, it may be desirable to operate in pressure equip ment when using other volatile organic amines as would be well-known to those skilled in the art.

The reaction of this invention comprises mixing 5,1 0-methenyl-5,6,7,8-tetrahydrofo- lic acid (anhydroleucovorin), an amine base, and water and heating the mixture at a temperature of from about 500-C to reflux for a period of time of from about 5-7 hours to give leucovorin. The pH of the mixture is adjusted to and maintained at 5 to 7 by the addition or removal, as appropriate, of suitable amounts of the amine bases useful in this invention. The anhydroleucovorin may be used in the form of its internal salt (i.e. zwitterion) or as a salt with a suitable anion, preferably chloride, normally as a hydrate. After the ring opening reaction is complete, the reaction mixture is generally cooled and filtered. Slightly more than an equlmolar (to anhydroleucovorin) amount of water soluble calcium saft, e.g. chloride, bromide, iodide, nitrate, acetate, is then added either as an aqueous solution or as a solid to the filtrate and calcium leucovorin is precipitated by adding a water soluble organic solvent, e.g. a lower alkanol such as methanol, ethanol, isopropanol, or acetone.

The calcium leucovorin is then removed by filtration, washed with a suitable solvent such as aqueous ethanol, ethanol, acetone, ether, ethyl acetate or the like and dried.

Column chromatography is generally not required since the product so obtained meets or exceeds USP specifications.

Example I 5,10-Methenyl-5,6,7,8-tetrahydrofolic acid, chloride hydrochloride dihydrate (10 g) was added in one portion to a mixture of 40% methylamine (6-5 ml) and water (90 ml) at 60"C. The mixture was heated to reflux and the pH adjusted to 6-0 by distilling out excess base. During the following 4 hour reflux period, the pH was maintained between 5.9 and 6-1 by addition of 40% methylamine. The mixture was cooled to room temperature, slurried with 10 g synthetic magnesium silicate and filtered. The filtrate was stored at --5"C for 16 hours, brought to room temperature and 2-25 g calcium chloride added. The product was precipitated by addition of 30 ml ethanol and collected by filtration. The cake was washed with 50% aqueous ethanol and with ether and dried under reduced pressure.

Yield 5.9 g (55%).

hplc analysis-931 %.

USP (Vol. XIX) Quantitative tlc assay for Calcium leucovorin-96 and 101%.

Example 2 5.10-Methenyl-5,6,7,8-tetrahydrofolic acid,.

chloride hydrochloride dihydrate (20 g) was added in one portion to a stirred mixture of triethvlamine (20 ml) and water (100 ml) at 65"C. The mixture was heated to reflux and the pH adiusted to 6-0 by distilling out excess base. The pH was maintained bo tween 5'6 and 6-1 for a 5 hour reflux period by addition of triethylamine. The mixture was stirred at room temperature for 16 hours, slurried for 30 minutes with 15 g synthetic magnesium silicate and filtered.

Calcium chloride (4 g) was added to the filtrate. The mixture was cooled in an ice bath and 40 ml ethanol was added. The precipitate was filtered, washed with 40 ml 50% aqueous ethanol, 30 ml ethanol, 30 ml ether and dried under reduced pressure.

Yield 121 g (57%).

hplc analysis--945%.

USP (Vol. XIX) Quantitative tIc assay for Calcium leucovorin96-4 and 983%.

Example 3 5,1 0-Methenyl-5,6,7,8-tetrahydrofolic-acid, chloride hydrochloride hydrate (4 g) was suspended in H2O (35 ml). The pH of the solution was adjusted to 65 by addition of appropriate amounts of morpholine. The mixture was maintained at reflux for 6 hours, cooled, slurried for 20 minutes with synthetic magnesium silicate and filtered.

The filtrate was adjusted to pH 8-0 with morpholine and aqueous calcium chloride added (0 9 g in 4 ml). The resultant solution was diluted with ethanol until cloudy and cooled at --5"C for one hour. The solid was filtered, washed with ethonal (15 ml), ether (20 ml) and dried under reduced pressure.

Yield 243 g (55 Ó).

hple analysis-83 %.

Further dilution of the original mother liquor with ethonal provided a second crop of solid product.

Yield 0-87 g (20%).

hplc analysis83%.

Example 4 5,10-Methenyl-5,6,7,8-tetrahydrofolic acid, chloride hydrochloride dihydrate (10 g) was added in one portion to a mixture of 20% tetraethyl-ammonium hydroxide (20 ml) and water (100 ml) at 50 C. Additional base. (30 ml) was added to adjust the pH to 6. The mixture was maintained at reflux for 4 hours with the pH kept between 5.5 and 6-0 by addition of tetraethylammonium hydroxide. The pH was adjusted to 6 35 and reflux continued for an additional 16 hours. The mixture was cooled and stirred at room temperature for 24 hours. Synthetic magnesium silicate (15 g) was added, slurried for 30 minutes and filtered off.

The solution was diluted to 150 ml with water, pH adjusted to 8-7 with tetraethylammonium hydroxide and SD3A added (50 ml). Calcium chloride (2 g) in a minimum amount of water was dropwise added to the solution. The resultant precipitate was filtered at SOC, washed with 50% aqueous SD3A (50 1) and slurried in 200 ml 0-1% tetraethylammonium hydroxide in SD3A for 1 hour. The solid was filtered, washed with acetones and dried under reduced pressure.

Yield 4 g (38%).

hplc analysis-94 4%.

Example S 5,10-Methenyl-5,6,7,8-tetrahydrofolic acid, chloride hydrochPsride dihydrate (20 g) was added in one portion to 100 ml water at 60"C followed by N,N-diethylethanolamine (14-9 g) which adjusted the pH to 6. The mixture was maintained at reflux for S hours and the pH kept between 5 7 and 6'2 by addition of N,N-diethylethanolamine.

The mixture was cooled, synthetic magne- sium silicate (15 g) added and slurried, and filtered through celite [Registreded Trade Mark and diluted with 40 ml SD3A. The filtrate was kept at --5DC for 16 hours, aqueous calcium chloride (4 0 g) was added dropwise to the sold filtrate, and the precipitate filtered, washed with SD3A (100 ml) and with ethyl acetate (100 ml) and dried under reduced pressure.

Yield 13 g (61%).

USP (Vol. XIX Quantitate tic assay for Calcium leucovorin 91%.

WHAT WE CLAIM IS:--- 1. The method of preparing calcium leucovorin which comprises the steps of mixing together anhydroleucovorin, an amine base, and an aqueous solvent, heating the mixture at a pH of 5-7, adding to the mixture an amount of a water-soluble calcium salt substantially equimolar to anhydroleucovorin, and isolating calcium leucovorin.

2. The method of claim 1 wherein the amine base is selected from ammonia, methylamine, dimethylamine, triethylamine, morpholine, trimethylamine, tetraethylammonium hydroxide, N,N-diethylethanolamine, aniline, p-chloroaniline, n butylamine, cyclohexylamine, diethanolamine, triethanolamine, diethylamine, piperidine, N,N-dimethylaniline, piperazine, Nmethylpiperazine, N,N'-dimethylpiperazine, pyridine, imidazole, benzylamine, ethylenediamine, o-toluidine"Zz-toluidine, ptoluidine, o-anisidine, m-anisidine, and ptanisi- dine.

3. The method of claim 1 wherein the amine base is selected from ethylamine, triethylamine, morpholine, tetraethylammonium hydroxide, and N,N-diethylethanolamine.

4. The method of claim I wherein the organic base is selected from methylamine, triethylamine, and N,N-diethylethanolamine.

5. The method of preparing leucovorin which comprises the steps of mixing together anhydroleucovorin, an amine base and an aqueous solvent and heating the mixture at a pH 5-7.

6. The method of claim S wherein the amine base of claim 2 is used.

7. The method of claim 5 wherein the amine base of claim 3 is used.

8. The method of claim 5 wherein the amine base of claim 4 is used.

9. The method of claim 1 or 5 wherein the amount of base used is sufficient to produce a pM of S to 7 in the mixture.

10. The method of preparing calcium leucovorin substantially as hereinbefore described with reference to any of the examples.

**WARNING** end of DESC field may overlap start of CLMS **.

Claims (10)

**WARNING** start of CLMS field may overlap end of DESC **. acetones and dried under reduced pressure. Yield 4 g (38%). hplc analysis-94 4%. Example S 5,10-Methenyl-5,6,7,8-tetrahydrofolic acid, chloride hydrochPsride dihydrate (20 g) was added in one portion to 100 ml water at 60"C followed by N,N-diethylethanolamine (14-9 g) which adjusted the pH to 6. The mixture was maintained at reflux for S hours and the pH kept between 5 7 and 6'2 by addition of N,N-diethylethanolamine. The mixture was cooled, synthetic magne- sium silicate (15 g) added and slurried, and filtered through celite [Registreded Trade Mark and diluted with 40 ml SD3A. The filtrate was kept at --5DC for 16 hours, aqueous calcium chloride (4 0 g) was added dropwise to the sold filtrate, and the precipitate filtered, washed with SD3A (100 ml) and with ethyl acetate (100 ml) and dried under reduced pressure. Yield 13 g (61%). USP (Vol. XIX Quantitate tic assay for Calcium leucovorin 91%. WHAT WE CLAIM IS:---

1. The method of preparing calcium leucovorin which comprises the steps of mixing together anhydroleucovorin, an amine base, and an aqueous solvent, heating the mixture at a pH of 5-7, adding to the mixture an amount of a water-soluble calcium salt substantially equimolar to anhydroleucovorin, and isolating calcium leucovorin.

2. The method of claim 1 wherein the amine base is selected from ammonia, methylamine, dimethylamine, triethylamine, morpholine, trimethylamine, tetraethylammonium hydroxide, N,N-diethylethanolamine, aniline, p-chloroaniline, n butylamine, cyclohexylamine, diethanolamine, triethanolamine, diethylamine, piperidine, N,N-dimethylaniline, piperazine, Nmethylpiperazine, N,N'-dimethylpiperazine, pyridine, imidazole, benzylamine, ethylenediamine, o-toluidine"Zz-toluidine, ptoluidine, o-anisidine, m-anisidine, and ptanisi- dine.

3. The method of claim 1 wherein the amine base is selected from ethylamine, triethylamine, morpholine, tetraethylammonium hydroxide, and N,N-diethylethanolamine.

4. The method of claim I wherein the organic base is selected from methylamine, triethylamine, and N,N-diethylethanolamine.

5. The method of preparing leucovorin which comprises the steps of mixing together anhydroleucovorin, an amine base and an aqueous solvent and heating the mixture at a pH 5-7.

6. The method of claim S wherein the amine base of claim 2 is used.

7. The method of claim 5 wherein the amine base of claim 3 is used.

8. The method of claim 5 wherein the amine base of claim 4 is used.

9. The method of claim 1 or 5 wherein the amount of base used is sufficient to produce a pM of S to 7 in the mixture.

10. The method of preparing calcium leucovorin substantially as hereinbefore described with reference to any of the examples.