AU619124B2 - Salts of n5,n10-methylene-5,6,7,8-tetrahydrofolic acid - Google Patents

Abstract

There are described salts of N<5>,N<10>-methylene-5,6,7,8-tetrahydro- folic acid, of the formulae Ia and Ib, <IMAGE> in the form of the (6RS) diastereoisomer mixture or in the form of the individual (6R) or (6S) diastereoisomers. The substituents are defined in Claim 1. There are also described three processes for the preparation of these salts. The abovementioned salts of the formulae Ia and Ib can be used for producing a pharmaceutical to act as a synergist with a compound for combating cancer, and/or for reducing the toxicity of a compound for combating cancer, and/or for protecting human and/or animal cells.

Description

_i i uhr-urr~,~---l~il^rrdrl- llll-UI 619124 COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952 COMPLETE SPECIFICATION NAME ADDRESS OF APPLICANT: Sapec S.A. fine chemicals Barbengo Lugano CH-6903 Switzerland NAME(S) OF INVENTOR(S): Fabrizio MARAZZA Attillo MELERA Rene VITERBO S. Nando TODERI ADDRESS FOR SERVICE: DAVIES COLLISON Patent Attorneys 1 Little Collins Street, Melbourne, 3000.

S COMPLETE SPECIFICATION FOR THE INVENTION ENTITLED: Salts of N 5

N

1 0 -methylene-5, 6, 7, S-tetrahydrofolic acid The following statement is a full description of this invention, including the best method of performing it known to me/us:- L I 0 il r' 1A i Field of the Invention The present invention is directed to salts of

N

5

,N

1 0 -methylene-5,6,7,8-tetrahyd-ofolic acid, processes for their preparation as well as to their use for the preparation of a medicament for the synergistic excertion of influence of a cancer controlling compound and/or for the reduction of the toxicity of a cancer controlling compound and/or for the protection of human and/or animal cells.

S' Background of the Invention toot

SN

5

,N

1 0 -Methylene-5,6,7,8-tetrahydrofolic acid of the formula II (abbreviated with CH 2 -THF) is the chemical species which acts as co-factor in the inhibition of the enzyme thymidilate synthetase (abbreviated with TS) together with 5-Fluorouracil (abbreviated with and/or one of its pro-drugs, such as Floxuridine, Tegafur. 5-FU or one of its pro-drugs are metabolically transformed into 5F-desoxyuridine monophosphate (abbreviated with 5F-dUMP), which is covalently and irreversibly bound to TS in the presence of CH 2 -THF, whet reby a ternary complex is formed, which is described as 2

-THF.

5-FU and its pro-drugs are widely used as che- 25 motherapheutic agents in the treatment of colon, rectal and pancreatic carcinoma, as well as other forms of cancer, such as breast and gastric cancer. In order to enhance the cytotoxicity for the tumorcells, the highest inhibition rate of TS must be achieved. This requires apropriate supply to those cells of both 5-FU (or one of i 2 its pro-drugs) and the co-factor CH 2 -THF. Whereas (or its pro-drugs) are exogeneous compounds, which can be supplied to patients in any desirable amounts, CH 2 THF originates normally from the endogeneous folate-pool via a number of metabolic transformations. However, the amount of CH 2 -THF, formed in this way, might not be sufficient for an efficient and complete inhibition of TS.

It has therefore become customary to use clinical treatment protocols, in which calcium-folinate (abbreviated with CaF), a metabolic precursor of

CH

2 -THF, is supplied in high doses to the patient simultaneously with 5-FU. See R.J. DeLap, The Yale Journal of Biology and Medicine, 61, 23 (1988). These high doses of CaF result in side effects, such as diarrhea and stomatitis; see G. Marini et al. Oncology 44, 336 (1987). The supply to the patient of adequate amounts of CH 2

-THF,

i.e. of the ternary complex forming species itself, could not be performed up to now, because no process for the large-.scale production of a sufficiently pure, easily soluble in water and stable compound was available.

Description of the Prior Art The preparation on a microscale of CH 2 -THF as the free acid has been described by several authors; see o .0 25 R.L. Blakley, Biochem. Journal, 72., 707 (1959); M.J.

Osborn, P.T. Talbert, F.M. Huennekens, J.Am. Chem. Soc.

0 82, 4921 (1960); R.G. Kallen, W.P. Jenks, J.Biol. Chem.

241, 5851 (1966); P.R. Farina, L.J. Farina, S.J.

Benkovic, J.Am. Chem. Soc. 95, 5409 (1973); R.P. Leary, Y. Gaumont, R.L. Kisliuk, Biochem. Biophys. Res. Comm.

56, 484 (1974); R.L. Kisliuk, D. Strumpf, Y. Gaumont, R.P. Leary, L. Palnte, J. Med. Chem. 20, 1531 (1977); ii Y D-Y-na~*rrmsmc 3 M. Poe, L.M. Jackman, S.T. Benkovic, Biochemistry 18, 5527 (1979) and R. Kalbermatten, W. Staedeli, J.H.

Bieri, M. Viscontini, Helv. Chim. Acta 64, 2627 (1981).

C. Zarow, A.M. Pellino, P.V. Daneberg, Prep.

Biochem. 12, 281 (1983) describes a "large scale" method of preparation, which involves an enzymatic reduction step.

However, in all cases the product could not be isolated otherwise than in a lyophilized form or as an amorphous powder (see C.M. Tatum Jr., P.A. Benkovic, S.T. Benkovic, R. Potts, Biochemistry 16, 1903 [1977]) °oo 0 and of a purity of not higher than 80 which make oo these products unadequate for a therapeutic use in humans.

O 0 0 oo 15 Two methods of synthesis have been described a a for obtaining CH 2 -THF as the free acid.

First process 0 0 5,6,7,8-tetrahydrofolic acid of the formula IV o COOH 0 000 20 H S o JI I (IV) H L H 25 COOH 'z I is reacted with an excess of formaldehyde in aqueous so- 4 lution at a pH-value of 7. See M. Poe, L.J. Jackman, S.T. Benkovic, Biochemistry 18, 5527 (1979); R. Kalbermatten, W. Staedeli, J.H. Bieri, M. Viscontini, Helv.

Chim. Acta 64, 2627 (1981); C. Zarow, A.M. Pellino, P.V. Danenberg, Prep. Biochem. 12, 381 (1983).This process is only suitable for the preparation of milligram-amounts of the free acid of the formula II 0 /COOH 0 H o

I

I)

00 15 N i H 0I

H

0 oo0

H

0 u 0 00 o0 0 020 0 0 0 25 and leeds to a product of low purity (less than 80 because of the inherent instability of CH 2 -THF in aqueous solution at pH 7; see R.G. Kallen, W.P. Jenks, J. Biol. Chem. 241, 5851 (1966).

.'i i") Second process

N

5

,N

10 -Methenyl-5,6,7,8-tetrahydrofolic acid in the form of its chloride of the formula V

H

H

CoH coovi is reduced in an anhydrous solvent with NaBH 4

CH

2

-THF

as the free acid of the above mentioned formula II is isolated as an amorphous solid with a purity from 75 to 80 see P.R. Farina, L.J. Farina, S.J. Benkovic, J. Am. Chem. Soc. 95, 5409 (1973); C.M. Tatum Jr., P.A. Benkovic, S.T. Benkovic, R. Potts, Biochemistry 16, 1903 (1977). CH 2 -THF in the form of the free acid is lua te.r practically insoluble in4wa;;ear, and as such it is unsuitable for parenteral pharmaceutical preparations.

Quite surprisingly were found processes, which are suitable for the large-scale production, for the preparation of new, stable and therapeutically acceptable watersoluble salts of CH 2 -THF, which are sufficiently pure to be administered to a patient.

Summary of the Invention Hence, it is a general object of the present invention to provide novel salts of N5,N 10 -methylene- 5,6,7,8-tetrahydrofolic acid.

It is a further object of the present invention to provide processes for the preparation of these salts.

It is still a further object of this invention to provide a medicament containing these salts.

These salts may be used&fhbz he praparatio ef a- mdidgant for the synergistic excertion of influence of a cancer controlling compound and/or for the reduction of the toxicity of a cancer controlling compound and/or for the protection of human and/or animal cells.

The present invention is characterized by the characteristics as contained in the independent claims.

0o Preferred embodiments are defined in the dependent claims.

0oo ao Q £0i0.

I 4 Detailed Description of the Preferred Embodiments In table I are summarized some preferred compounds of this invention.

JL4 H )R

H--H

H coo R Ito I lilt lit I 091)0 0 0 00 00 0 I 0 0 000 3 a R H known ~b R =Na 4 ,Pyridinium TE R =,[HO(CH2)2NH3]+ T_ R {HO(CH2)2]3NH)+ 3e R [HO(CH2)2NH(CH3)21+ TfR r(G1-1) 3 CNH3]+ 3h R -Arg in ine] 3i R =1/2 Ca 2 3k R =1/2 Mg 2 T1 R Na+ 3m

R+

Rn R Li+ 0 00 0 010 0 0 In table II is shown schematically the preparation of some preferred compounds of this invention.

8 Table II H O R-W-

-H

H--H

H -H

H+

SHSO+

000 0 00 0Q0 0. 0 0 00 0.0 0 00.0 0 0 0 00 00 0 00 00 H COH

H--H

H--H

CO ON or 2 Me+ 0WO (Method A) H Coo- PI-42+ R-W H H H H tH COO- R-NHj o r 1+ 3 c-L 1 e-n 1. No OH

IC

Lt?~ H Coo- Coo- Coo- (M~ethod B) Us~2n R= N 5

N

10 -Methylele-5 8-tetrahydropteroyl- 9 The various salts of this invention may be prepared either directly from the compound of the formula 3a (method A) or by the reaction of a alkaline earth metal salt, for example the calcium salt of the formula 3i, with the oxalic acid salt of the corresponding cation or amine (method B).

one S0 0 00 0 0 0 0 000 0 0 0 00o 4 0 00 00 0 U 'a 3 O 0 .i 00 0 000 0 0 00 0 0 tt 0 0 0 0 000 oo 0 4) 0 0 0 00 00 0 o 0 The HPLC purity of the CH 2 -THF salts, obtained in this way, varies between 83.8 and 92.4 the major "contamination" of this preparation consists of the well known and therapeutically useful (6RS)-N 5 -Methyl- 5,6,7,8-tetrahydrofolic acid, so that the total amount of reduced folates in the preparation is on the average 95 Method A 15 The compound of the formula 3a was prepared by reacting the compound of the formula V in a solvent, such as DMSO (dimethylsulfoxide) in the presence of a base, such as pyridine, with the respective amount of NaBH 4 whereby one obtains the compound of the formula 20 3b, which is further dissolved in a cold KOH-solution; the compound of the formula 3a is obtained by acidification.

The compound of the formula 3a is successively reacted with a pharmaceutical acceptable base, such as ethanolamine, triethanolamine, 2-dimethylaminoethanol, tert.-butylamine, or with amino acids, such as lysine, arginine, whereby watersoluble salts are formed. The compound of the formula 3a may also be reacted with catins, such as Ca 2 Mg 2 Li+, Na+, whereby the corresponding watersoluble salts are formed, which are isolated by precipitation from the aqueous solution by the addition of an organic solvent, such as ethanol, acetone. See table II.

Method B The alkaline earth metal salts, for example the compound of the formula, 3i, which were obtained by method A, were reacted with the oxalic acid salt of the selected amine or the corresponding amino acid or the corresponding cation. Due to the precipitation of the waterinsoluble alkaline earth metal oxalate the correspondingly derived salt remains in solution, and is only then precipitated by the addition of an organic waterso- 0 )00 0o 0 luble solvent, such as ethanol, acetone. See table II.

o oe Method C ooo Sa° Quite surprisingly was found a still further, 0 0 o' 15 third process, which is suitable for the large scale production, for the preparation of alkaline earth metal salts of N 5

,N

10 methylene-5,6,7,8-tetrahydrofolic acid.

0 0, (i o O With the third inventive method the alkaline ooe o earth metal salts are obtained in high yields and with S 20 high purity. Additionally this inventive method is car- 0 o ried out in an aqueous medium, what simplifies extremely the isolation of the desired alkaline earth metal salts.

o In this inventive method is submitted an o *3 aqueous solution of a watersoluble salt of the 5,6,7,8tetrahydrofolic acid, and then this one is reacted with formaldehyde. Then is added an aqueous solution of an alkaline earth metea salt, whereby the corresponding alkaline earth metal Salt precipitates.

The calcium salt and the magnesiul, salt of

N

5

,N

1 0 -methylene-5,6,7,8-tetrahydrofolic acid are preferred.

The 5,6,7,8-tetrahydrofolic acid may be prepared for example according to the teachings of E. Khalifa, A.N. Ganguly, J.H. Bieri and M. Viscontini, Helv. Ch. Acta (1980), 63, 2554.

The inventive salts may be used for the preparation of a medicament for the synergistic excertion of influence of a cancer controlling compound and/or for the reduction of the toxicity of a cancer controlling 0o0 0 compound and/or for the protection of human and/or anio c, mal cells. Such a medicament contains usually at least one active salt in an amount from 1 mg to 500 mg, especially from 5 mg to 150 mg, per dosis unit. Preferrably oo the medicament is in the form of a parenteral and/or o oral pharmaceutical preparation.

The following examples shall illustrate the invention. Clinical results and therapeutical applica- S 20 tions of these compounds are also described.

,o Examples Example 1 Preparation of the (6RS)-N 5

,N

10 -methylene- 5,6,7,8-tetrahydrofolic acid of the formula 3a.

To a solution of 24.6 g of the compound of the formula V (prepared according to C. Temple, R.D. Elliot, J.D. Rose, J.A. Montgomery, J. Med. Chem. 22, 731 [1979]) in a mixture of 300 ml dry DMSO and 100 ml dry L-il~~ Jr_~l~LU I I- I~ ii 12 pyridine, stirred under the exclusion of oxygen at room temperature, a solution of 2.0 g NaBH 4 in 100 ml DMSO was added dropwise during one hour. This mixture was then diluted with two litres of acetone during a period of 20 minutes. After an additional stirring of the mixture during one hour at a temperature of the precipitated mixed salt of the formula 3b was collected by filtration, and washed three times with fresh acetone.

After the drying at reduced pressure 16.17 g of the raw compound of the formula 3b were isolated.

The HPLC analysis showed, that this product was 87.1 pure.

o The above mentioned product was added to 1 420 ml degassed water, and stirred under the exclusion of oxygen at a temperature of 0.5 M cold KOH was then added dropwise to the stirred suspension, until a Scomplete dissolution was observed. The pH-value of the so obtained solution was 7.5, and the free acid of the formula 3a was precipitated by means of the dropwise addition of cold 1N HC1, until the pH-value of the supernatant reached a value of 3.95. After an additional stirring at +4°C during 10 minutes the free a.id of the fcrmula 3a was collected by filtration, washed once with cold water, as well as once with acetone and once with peroxide free diethylether.

After drying at reduced pressure 11.38 g of the amorphous compound of the formula 3a were isolated.

HPLC analysis: 86.5 purity 7.6 _i, 1 to b.

YT 13 This product was not purified further but converted into several salts, as described below.

Example 2 Preparation of the ethanolamine salt of (6RS)-

N

5

,N

10 methylene-5,6,7,8-tetrahydrofolic acid of the formula 3c (method A).

To a solution of 11.36 g of the comrpound of the formula 3a, dissolved in 170 ml dry DMSO and stirred at room temperature under the exclusion of oxygen, a solution of 3.0 ml ethanolamine (2 equivalents) in 50 ml DMSO was added dropwise. The mixture was then freed by filtration from a slight turbitity and added slowly to 600 ml stirred ethylacetate. After an additional stirring during 20 minutes at room temperature the precipitated product was collected by filtration and washed twice with ethylacetate and once with diethylether. After drying at reduced pressure 14.22 g of the compound of the formula 3c were isolated.

0900 0co" HPLC-analysis: 83.8 purity 7.1 0 oo 20 N 5 -methyl-THF.

00 0 0o 0 "0 UV (c 20 mg/i in 0.1 M phosphate-buffer at pH 7.3): Amax 293 nm E 43000) Amin 247 nm Amax Amin 3 9 [a]D 10.4" (c 1 in 0.1 M phosphate-buffer at pH 7.3).

14 NMR (220 MHz in DMSO-pyridine 9:1) 2 characteristic doublets (J 4 Hz) at 5.05 ppM (1H) 3.85 ppM (1H) for the C-1 methylene protons.

Example 3 Preparation of the triethanolamine salt of (6RS)-N 5 ,N10-methylene-5,6,7,8-tetrahydrofolic acid of the formula 3d (method A).

To a solution of 4.35 g of the compound of the formula 3a in 50 ml dry DMSO, stirred at room temperature under argon, a solution of 2.84 g triethanolamine (2 equivalents) in 14 ml DMSO was added dropwise. After O o filtering off a small amount of a white precipitate, the oo onu solution was added dropwise to a stirred mixture of so 220 ml ethanol and 110 ml diethylether.

o a o 1 o 15 The suspension was stirred for further 30 minutes at a temperature of The solid was collected by filtration, washed once with ethanol/diethylether 2:1, and once with diethylether, and dried at reduced o.o° pressure, whereby 4.88 g of the compound of the formula So 20 3d were obtained.

0 a i 5 HPLC-analysis: 90.3 purity 5.8 TV (c 20 mg/l in 0.1 M phosphate-buffer at S.A pH 7.3): limx 295 nm (E 37800) Amin 246 nm Amax Amin 4.9.

.I .e L II~ I i -j 1 P~P a+asrsr~l-r -larsl r~ss~ [a]D 11.7" (c 1 in 0.1 M phosphate-buffer at pH 7.3).

NMR (220 MHz in D 2 0/NaOD): 2 characteristic doublets (J 4 Hz) at 5.03 ppM (1H) 3.90 ppM (1H) for the C-13 withylene protons.

Example 4 Preparation of the 2-dimethylaminoethanol salt of (6RS)-N5,N10-methylene-5,6,7,8-tetrahydrofolic acid of the formula 3e (method A).

10 To a solution of 4.0 g of the compound of the "o formula 3a in 46 ml dry DMSO stirred at room temperature o o '0o0 under the exclusion of oxygen, a solution of 1.76 ml 0 2-dimethylaminoethanol (2 equivalents) in 13 ml DMSO was 000 added tropwise. After filtering off from a small amount "o 15 of precipitate, the solution was added dropwise to a stirred mixture of 200 ml ethanol and 100 ml diethylether at a temperature of After stirring for further 30 minutes at the same temperature, the soooo lid was collected by filtration, and washed once with 000 20 ethanol-diethylether 2:1 and once with diethylether. After drying at reduced pressure 3.93 g of the compound of the formula 3e were isolated.

HPLC-analysis: 91 purity 5.7

THF.

UV (c 20 mg/1 in 0.1 M phosphate-buffer at pH 7.3): Amax 295 nm (E 31200) Amin 246 nm Amax Amin R.P. Leary, L. Painte, J. meu. unem. zu, .j \L ,1 I [a]D 12.1" (c 7.3).

1 in 0.1 M phosphate-buffer at pH doublets the C-11 NMR (220 MHz in (J 4 Hz) at 5.03 methylene protons.

D

2 0/NaOD): ppM (1H) 2 characteristic 3.90 ppM (1H) for ooo o o So o B 00 0 Example Preparation of the tert.-butylamine salt of (6RS)-N 5 ,N10-methylene-5,6,7,8-tetrahydrofolic acid of the formula 3f (method A).

To a solution of 4.3 g of the compound of the formula 3a in 60 ml dry DMSO, stirred at room temperature under argon, a solution of 1.97 ml tert.-butylamine (2 equivalents) in 17 ml DMSO was added dropwise. After a few minutes of stirring a thick precipitate was formed. The mixture was stirred further at a temperature of 18°C during one hour. The product was isolated by repeated centrifugation at 3500 rpm, resuspending it first in a mixture of 100 ml ethanol and 50 ml diethylether, and then in pure diethylether.

After drying at reduced pressure 4.63 g of the compound of the formula 3f were isolated.

HPLC-analysis: 89 purity 7.1

THF.

UV (c 20 mg/l in 0.1 M phosphate-buffer at pH 7.3): max 295 nm (E 32700) Amin 246 nm Amax Amin J: _CL.Lci:U WIL il cLI tc 2; t- UL I ULllICj-UtiyIUt LI1 CILU=LU. 17 [a]D 11.00 (c 1 in 0.1 M phosphate-buffer at pH 7.3).

NMR (220 MHz in D 2 0/NaOD): 2 characteristic doublets (J 4 Hz) at 5.03 ppM (1H) 3.90 ppM (1H) for the C-1 methylene protons.

Example 6 Preparation of the (L)-lysine salt of (6RS)-

N

5 ,N1 0 -methylene-5,6,7,8-tetrahydrofolic acid of the formula 3g (method A).

Oo 10 To a solution of 4.05 g of the compound of the o" formula 3a in 55 ml dry DMSO, stirred at room tempera- 00 0 ture under argon, a solution of 2.60 g (L)-lysine oo (2 equivalents) in 15 ml DMSO was added dropwise. After o the stirring during a few minutes a thick precipitate 0 00 00 15 was formed. The mixture was stirred further at a temperature of 18 0 C during one hour. The product was isolated by repeated centrifugation at 3500 rpm, resuspending it first in a mixture of 100 ml ethanol and 0o"0 50 ml diethylether and then in pure diethylether.

0 00 After drying at reduced pressure 5.03 g of the °compound of the formula 3g were isolated.

HPLC-analysis: 90 purity 6.1

THF.

UV (c 20 mg/l in 0.1 M phosphate-buffer at pH 7.3): Amax 295 nm (E 29500) Amin 246 nm Amax Amin 4.4.

111..1~ c A PI L- CILIN g lli--r~-ll-~ [a]D 11.5" (c 1 in 0.1 M phosphate-buffer at pH 7.3).

NMR (220 MHz in D 2 0/NaOD): 2 characteristic doublets (J 4 Hz) at 5.03 ppM (1H) 3.90 ppM (1H) for the C-ll methylene protons.

Example 7 Preparation of the (L)-arginine salt of (6RS)- N5,N0-methylene-5,6,7,8-tetrahydrofolic acid of the formula 3h (method A).

1. 0 The (L)-arginine salt of the formula 3h was prepared in analogous way as described in example 6.

Example 8 Preparation of the calcium salt of (6RS)-

N

5

N

1 0 -methylene-5,6,7,8-tetrahydrofolic acid of the formula 3i (method A).

g of the raw compound of the formula 3b was added in small portions to a mixture of 16 ml degassed water and one drop of lN NaOH, and stirred vigorously at a temperature of During the addition the pH-value of the mixture was kept at about 9.0 through the occasional addition of IN NaOH. Then 0.92 ml of a 2M calcium chloride solution was added to the mixture. After the stirring during 10 minutes the solution was freed from an insoluble precipitate by means of a filtration. Finally, the mixture was diluted dropwise with ml acetone unter stirring at a temperature of +4°C, whereby a yellowish precipitate was obtained, which was isolated by filtration, and washed once with absolute J I I I 19 ethanol and once with acetone and dried at reduced pressure. In this way 673 mg of the calcium salt of the f ormula 3i were isolated.

HPLC-analysis: 91 purity 6% UV (c 20 mg/l in 0.1 M phosphate-buffer at pH 7.3): Amax 296 nm (C 28900) AXmin 245 nm Amax Amin 3.68.

[a]D 12.6' (c 1 in 0.01 N Na0H).

0000 0 00 00 0 0 00 00 0 000 0 0 00 0 0 0 0.1 0 0 0000 0 00 00 0 0 00 00 0 000 0

H

2 0 content (according to Karl Fischer): 8.9 Elemental analysis for CH 2 -THF.Ca X 3.5 H120 calculated value 43.02 exp. value 43.27 0 0 00 0 0 ~0-0 0 0 5 .05 17.56 4.84 17.57 6.9,9. %Ca 7.18 Example 9 Preparation of the magnesium salt of (6RS) N 5

,N

1 0 methylene-5,6,7,8-tetrahydrofolic acid of the formula 3k (method A).

L_ g of the raw compound of the formula 3b was added in small portions to a mixture of 16 ml degassed water and one drop of IN NaOH, and stirred vigorously at a temperature of 4*C. During the addition the pH-value of the mixture was kept at about through the occasional addition of lN NaOH. Then 0.92 ml of a 2M magnesium chloride solution was added to the mixture. After stirring during 10 minutes the solution was freed from a little 'isoluble precipitate by filtration. The mixture was dilated dropwise with 20 ml acetone, whereby the mixture was stirred at a temperature of 4 0 C. A yellowish precipitate was obtained, which was isolated by filtration, and washed once with H 2 0, o once with ethanol, and once with acetone, and dried at 0 f o"o 15 reduced pressure. In this way 537 mg of the magnesium salt of the formula 3k were isolated.

HPLC-analysis: 92.4% purity 3.4% 0 THF.

UV (c 20 mg/l in 0.1 M phosphate-buffer at pH 7.3): °0 Amax 296 nm (E 27600) Amin 244 nm Amax Amin 3.68 [L]D 12.1° (c 1 in 0.1 N NaOH).

o°.0 25 H20 content (according to Karl Fischer): 8.9 o o0 4

''II

u Elemental analysis for CH 2 -THF.Mg x 5 H 2 0 calculated value 42.17 5.49 17.21 4.27 exp. value 42.26 5.51 16.30 5.02. %Mg Sample dried in the pentahydrate.

air: 15.8 H20 0 0 o0 0 0 00 0 o 0 00) 0 0.1 0 0 0 0 0 00 0 0 0 6.04 o o o ao 0 0 00 00 0 00 0; 0 Example Preparation of the sodium salt of (6RS)-

N

5

,N

1 0 methylene-5,6,7,8-tetrahydrofolic acid of the formula 31 (method B).

1.0 g of the calcium salt of the formula 3i was added to 15 ml of a 0.133 M solution of sodium oxalate in H 2 0, and stirred at a temperature of +4°C under the exclusion of oxygen. After 30 minutes stirring at the same temperature the precipitated calcium oxalate was removed by centrifugation from the mixture, and a clear supernatant was obtained, which was diluted slowly 20 with 75 ml acetone under stirring at a temperature of +4 0 C. The obtained precipitate was isolated by filtration, washed once with fresh acetone and dried at reduced pressure, to yield 737 mg of the compound of the formula 31 as a yellow powder.

HPLC-analysis: 88.6% purity 4.5% N 5 methyl-

THF.

I 1_1_ ~1_1~ UV (c 20 mg/1 in 0.1 M phosphate-buffer at pH 7.3): Amax 296 nm (E 29600) Amin 245 nm Amax Amin 4.00.

[a]D 12.5° (c 1 in 0.01 N NaOH).

Example 11 Preparation of the tert.-butylamine salt of (6RS)-N 5

,N

10 -methylene-5,6,7,8-tetrahydrofolic acid of the formula 3f (method B).

o 10 1.0 g of the calcium salt of the formula 3i Swas added to 15 ml of a 0.133 M solution of the oxalic 0 o O acid salt of tert.-butylamine, and was stirred at a temo o perature of +4"C under argon. After 30 minutes of stirring at the same temperature the precipitated calcium oxalate was removed by centrifugation, and the obtained clear supernatant was diluted slowly with 75 ml acetone under stirring at a temperature of The obtained precipitate was isolated by filtration, washed once with o~"oa fresh acetone and dried at reduced pressure, to yield 965 mg of the compound of the formula 3f as a yellow S powder.

HPLC-analysis: 87.2% purity 5.1%

N

5 -methyl-THF.

[Further analytical data: see example ii

I

Example 12 Preparation of the magnesium salt of N5,N0-methylene-5,6,7,8-tetrahydrofolic acid of the formula 3k (method C).

65 g of 5,6,7,8-Tetrahydrofolic acid were suspended in 400 ml water, and the pH-value was adjusted to a value of 9.0 by means of the addition of concentrated ammonia, whereby a clear solution was obtained. Then, 13 ml of a 36% aqueous formaldchyde solution were added dropwise, and the mixture was allowed to stand during minutes at room temperature.

2 o" After the addition of a solution of 50 g pon MgC1 2 .6H O in 50 ml water the so obtained mixture was stirred during 30 minutes at room temperature, and was O o 15 then allowed to stand during 2 hours. Then the desired o° product was filtered by suction, and washed once with water and once with ethanol. After the drying at reduced pressure at 50"C 27 g of the pure N 5,6,7,8-tetrahydrofolic acid magnesium salt were isola- 20 ted.

I HPLC-analysis: 98.8% purity.

SUV (c 20 mg/l in 0.01 N NaOH): Smax 296 nm (C 30200) Amin 245 nm Amax Amin 4.82.

[a]D (c 1 in 0.01 N NaOH).

I

I~

Example 13 Preparation of the calcium salt of (6RS)-

N

5

,N

10 -methylene-5,6,7,8-tetrahydrofolic acid of the formula 3i (method C).

190 g of 5,6,7,8-Tetrahydrofolic acid /are suspended in 1250 ml water. Then, 90 ml concentrated ammonia and 50 ml pyridine were added, whereby a pH-value of 8.5 was obtained.

After the addition of 40 ml aqueous 36 formaldehyde solution the mixture was filtered through a o<\oo layer of active charcoal and celite.

C -8a, To the obtained clear solution 200 ml of a 30 calciumchloride solution were added under stirring, and the pH-value was adjusted to a value of 9.0 by the addition of concentrated ammonia.

Then was cooled to a temperature of and the mixture was allowed to stand during 2 hours. Then was filtered off, and the obtained product was washed once with water and once with ethanol. After drying at reduced pressure at a temperature of 50°C 180 g of the pure N5,N 10 -methylene-5,6,7,8-tetrahydrofolic calcium salt were isolated.

HPLC-analysis: 96.5% purity 0.6%

THF.

UV (c 20 mg/l in 0.01 N NaOH): Amax 296 nm (E 26400) Amin 245 nm Amax Amin 4.87.

II-.

I

[a] D 17.3" (c 1 in 0.01 N NaOH).

and (6S)-N 5

,N

10 -methylene-5,6,7,8tetrahydrofolic acid salts according to the examples 2 to 11 were obtained in the same way, starting from the (6R) and (6S) free acids, respectively.

Stability of the compounds of the formulae 3c dr_ lR 3d 3e and 3f in 0.1 M phosphate-buffer 00 0 o 0 00 0 00 C 000 0 0 00 00 0 o o A 0.1 solution in 0.1 M phosphate-buffer/pH 10 9.0 of each of the title compounds were analysed by HPLC at the times 0, 60, 120, 300 minutes, whereby always 2.5u1 injections were made. The percentual recovery at the time x (Rx) was obtained by the following formula: Rx 100, whereby 15 A shows the integrated CH 2 -THF peak area at the time 0 and B shows the integrated peak area at the time x.

oo 0 0 0 00 o 41t o o time 0 Rx RX (3d) Rx (3e) Rx (.Lf 120 300 100 96.7 93.3 85.1 100 95.7 92.1 100 97.6 94.2 88.3 100 97.0 94.3 87.5 i

B

Pharmaceutical preparations for iniections a) Ampules with liquids The compounds of the examples 2 to 13, in an amount from 5 to 150 mg, were dissolved in a pharmaceutically acceptable buffer, such as 0.1 M phosphate-buffer, so that the solution had a pH-value of The solution was filtered up to sterility and fillet, in dark glas ampules, which were sealed under steril conditions.

Example A compound of the examples 2 to 13 5-150 mg; buffer solution, adjusted to pH 9.0 1-50 ml.

b) Lyophilized preparations The compounds of examples 2 to 13, in an amount from 50 to 250 mg, were dissolved either in a pharmaceutically acceptable buffer, such as 0.1 M phosphate-buffer or in a physiological sodium chloride solution. The solution was filtered up to sterility, filled in vials and lyophilized in a manner well known to those skilled in the art. When needed, the powder can be reconstituted by the addition of steril water prior to the administration.

i--Llli a.ylllb~-~ ~3 27 Example A compound of examples 2 to 13: 50 to 250 mg; either a buffer-solut:on or a physiological sodiumchloride solution: 3 to 20 ml.

Lyophilization of the sterile solution.

Pharmaceutical preparations for oral use As an example of an oral preparation formulations of enteric coated tablets are described, whereby a0,9 oo it is clearly understood, that further oral forms, such o°o 10 as enteric coated capsules may be prepared in an analo- Oo gous way.

o o Enteric coated tablets o o 0 The enteric coated tablets, which may contain from to 5 to 30 mg of a component of examples 2 to 13, are prepared in the following manner known per se in the art, whereby the entero-coating is performed according to "Remington Pharmaceutical Sciences", page 1614, edition, 1975 and "Theory and Practice of Industrial Pharmacy", Lackman, Liberman, Canig, pages 116, 371, 470 2nd edition 1976.

'N Example Compound of examples 1 to 13: 5 bis 30 mg Lactose USP: Starch NF: 150 bis 125 mg 35 bis 30 mg Magnesium stearate NF: Eudragit NF: Talc NF Propylene glycol 6000 NF: 3 mg 4 mg 4 mg 0,4 mg.

Clinical results and therapeutical applications of salts of N 5

N

10 -methylene-5,6,7,8-tetrahydrofolic acid As ex, ples for the possible therapeutical applications of e described compounds the following clinical results a e available: Patient A Sex: female Age: 19 Diagnosis: osteogenic sarcoma with respiration troubles and acataposis 29 Treatment: 5-Fluorouracil intravenously administered (300 750 mg/week) in combination with intravenously administered N 5

,N

10 -methylene-5,6,7,8tetrahydrofolic acid calcium salt (100 mg/week).

Clinical findings after a five weeks treatment: visible reduction of the tumor volume disappearance of the respiration troubles and of acataposis.

Patient B 0 0 0" 00 00 0 000 0 0 0 Q) id 0 .0 0 0 00 0 .3 0? 0 00 0 0 0 00 0 0 000 O 004* 0 €4 0.4 00044 oo 1 15 Sex: Age: Diagnosis: Treatment: 20 male 52 ive-r Rectum tumor with1Jvar metastasis 5-Fluorouracil intravenously administered (300 mg/week) in combination with intravenously administered

N

5

,N

10 -methylene-5,6,7,8-tetrahydrofolic acid calcium salt (100 mg/week).

Clinical findings after a five weeks treatment: Necrosis of the tumor and excretion of the necrotic tissue increase in weight of the patient.

It is noted that a previous treatment of the patient with equivalent amounts of 5-Fluorouracil and Leucovorin-calcium gave no notable amelioration of the clinical state.

_II 9 rl-C L- 0 Patient C Sex: Age: Diagnosis: Treatment: female: 53 Breast cancer left side 5-Fluorouracil intravenously administered (2 x 500 mg/week during 3 weeks and then (2 x 250 mg/week) during 2 weeks in combination with intravenously administered N 5

,N

1 0 -methylene-5,6,7,8tetrahydrofolic acid calcium salt (100 mg/5-Fluorouracil administration).

0 0 0 0 09 0 0 0o o 000 0 0 00 00 0 0 a Clinical findings after 8 weeks: 15 Tumor practically no longer visible or touchable Curing fibrotic scars.

The possible therapeutical applications are not restricted to the treatment of the above mentioned tumors with salts of the N5,N 1 0 -methylene-5,6,7,8tetrahydrofolic acid in combination with atHee are also treatments of further tumor kinds with salts of the N5,N0-methylene-5,6,7,8-tetrahydrofolic acid in combination with 5-Fluorouracil or further chemotherapeutica, such as for example Floxuridin (FUDR), Methotrexat, Cisplatin, Adriamycin, Vincristin, and so on.

While there are shown and described present preferred embodiments of the invention, it is to be distinctly understood, that the invention is not limited thereto, but may be otherwise variously embodied and practiced within the scope of the following claims.

1-a)O

Claims (29)

1. Salts of N 5 N 1 0 -methylene-5,6,7,8- tetrahydrofolic acid of the formulae Ia and Ib H A-N- H- H- H H H e COO COO R 1 H A-N H H -H H H coo R \R 000 0 00 000o 0 01, o&*0 0000 6 p 0 0 o 0* 00 0 000 0 **00 o 0 00.0 0 0 0 0 0 i" s in the form of the (6RS)-diastereoisomers mixture or in the form of the single (6R) or (6S) diastereoisomers, wherein 20 R is a pharmaceutically and/or therapeutically acceptable multivalent organic or inorganic cation, R 1 and R 2 are pharmaceutically and/or therapeutically acceptable monovalent organic and/or inorganic 25 cations, whereby R 1 and R 2 may be the same or be different, and A is a residue of the formula H )911028,dbdat089,59059.res,31 4** I LII-----^-C-LIYII _LII^III--~ -32-

2. Salts according to claim 1, characterized in that the inorganic cations are alkali metal cations or alkaline earth metal cations.

3. Salts according to claim 1 or claim 2, characterized in that the inorganic cations are selected from Li+, Na+, K Ca 2 Mg 2

4. Salts according to claim 1, characterized in that the organic cations are selected from the group consisting of ethanolamine, triethanolamine, 2- dimethylaminoethanol, tert.-butylamine and a basic amino so* acid. 0 0 0 o 15 5. Salts according to claim 4, characterized in ao that the basic amino acid is lysine or arginine.

6. Salts according to claim 4 or claim characterized in that the basic amino acid is (L)-lysine s 20 or (L)-arginine. O 0 000 0 0 00

7. A process for the preparation of salts 0*.o according to any one of claims 1 to 6, characterized in that N 5 ,N 1 0 -methylene-5,6,7,8-tetrahydrofolic acid of the 25 formula II COOH H 0 I3 0 I o o H H H COOH in the form of the (6RS)-diastereoisomers mixture or in the form of the single (6R) or (6S)-diastereoisomers is reacted with a pharmaceutically and/or therapeutically acceptable organic or inorganic base or with a salt of a ,911028,dbdat089,59059.res,32 '^ue' El~r ,IPC Oll i -33- pharmaceutically and/or therapeutically acceptable inorganic cation.

8. A process according to claim 7, characterized in that the reaction is carried out in at least one solvent.

9. A process according to claim 8, characterized in that the solvent is selected form dimethylsulfoxide, diemethylformamide, N,N-dimethylacetamide, N-methyl- pyrrolidone and water. so r o

10. A process for the preparation of salts o0e "av according to any one of claims 1 to 6, characterized in 15 that an alkaline earth metal salt of the formula III 0 0H 0 coo 0 44 H o 20 o620 H o o oxalic acid salt, whereby the alkaline earth metal oxalate precipitates, and whereby simultaneously the corresponding pharmaceutically and/or therapeutically acceptable organic and/or inorganic salt of the compound of the formula I is formed, then separating the precipitated alkaline earth metal oxalate, and then precipitating from the solution the desired salt of 911028,dbdat089,59059.res,33 I .1 L- i I L-O~----~IIP~FX-~Llsl~ ll~*~ -34- formula I by means of the addition of at least one water mixable organic solvent.

11. A process according to claim 10, characterized in that the alkaline earth metal cation is Ca 2 or Mg 2

12. A process according to claim 10 or 11, characterized in that the water mixable organic solvent is acetone, methanol or ethanol. .0

13. A process for the preparation of alkaline earth metal salts of N 5 ,N 10 -methylene-5,6,7,8-tetrahydrofolic acid of the formula I' rtl r r t: I t e coo" C- '\R ZN I 4 f~I in the form of the (6RS)-diastereoisomers mixture or in the form of the single (6R) or (6S)-diastereoisomers, wherein R' is an alkaline earth metal cation, characterized in that an aqueous solution of a soluble salt of the 5,6,7,8-tetrahydrofolic acid of formula IV cooH water the (IV) o H I, CoOH 911028,dbdat.089,59059.res,34 -1 1 is submitted and then reacted with formaldehyde, and then is added an aqueous solution of an alkaline earth metal salt, whereby the corresponding alkaline.earth metal salt of the formula I' precipitates.

14. A process according to claim 13, characterized in that the alkaline earth metal cation is Ca 2 or Mg 2

15. A process according to claim 13 or 14, oae characterized in that prior to the addition of formaldehyde a watersoluble, aromatic, nitrogen containing base is added. 0 0 0 oc

16. A process according to claim 15, characterized in that the nitrogen containing base is selected from pyridine and substituted pyridine.

17. A process according to claim 16, characterized in that substituted pyridine is selected from picoline, lutidine and ethylpyridine.

18. A process according to any one of claims 13 to 17, characterized in that the watersoluble salt of the 5,6,7,8-tetrahydrofolic ac'.d is prepared by reacting the free acid in water together with a compound selected from alkalihydroxides, alkalicarbonate, alkalihydrogen- carbonate and ammonia and organic, nitrogen containing bases.

19. A process according to claim 18, characterized in that the ammonia and organic, nitrogen containing bases are selected from pyridine, triethylamine and triethanolamine. A process according to any one of claims 13 to 19, characterized in that after the addition of the 911028,dbdaLO89,59059.res,35 -36- alkaline earth metal salt solution the pH-value i adjusted to a value in the range from 8.5 to

21. A process according to claim 20, characterized in that the pH-value is adjusted to a value of

22. A process according to claim 20 or 21, characterized in that the pH-value is adjusted by the addition of an aqueous ammonia solution.

23. A process according to any one of claims 13 to 22, characterized in that the reaction is carried out at \a temperature in the range from 10°C to 30 0 C. ""So 15 24. A process according to claim 23, characterized DO 4 o HoV in that the reaction is carried out at room temperature. 0*O 0 A process according to one of claims 13 to 24, characterized in that the magnesium salt of the N 5 ,N 10 20 methylene-5,6,7,8-tetrahydrofolic acid is prepared by o. reacting at room temperature the 5,6,7,8-tetrahydrofolic 0 0 0 acid in water with a compound, selected from 0 alkalihydroxides, alkalicarbonate, alkalihydrogencarbonate and ammonia and organic, nitrogen containing base, then reacting the so prepared salt solution with formaldehyde and then adding an aqueous solution of a magnesium salt, whereby the magnesium salt of the N 5 ,N 10 -methylene-5,6,7,8-tetrahydrofolic acid precipitates.

26. A process according to claim 25, characterized in that the nitrogen containing base is selected from pyridine, triethylamine and triethanolamine.

27. A process according to any one of claims 13 to 24, characterized in that the calcium salt of the N 5 ,N 10 methylene-5,6,7,8-tetrahydrofolic acid is prepared by i 911028,dbdat089,59059.res,36 -37- reacting at room temperature the 5,6,7,8-tetrahydrofolic acid in water with a compound, selected from alkalihydroxide, alkalicarbonate, alkalihydrogencarbon and ammonia and organic, nitrogen containing base, then adding to the so prepared salt solution a watersoluble, aromatic, nitrogen containing base, which is especially selected from pyridine and substituted pyridine. then reacting the so prepared salt solution with formaldehyde, and s t then adding an aqueous solution of a calcium salt, 15 whereby the calcium salt of the N 5 ,N 10 -methylene-5,6,7,8- o Oo tetrahydrofolic acid precipitates.

28. A process according to claim 27, characterized in that the nitrogen containing base is selected from oaao 20 pyridine, triethylamine and triethanolamine. o o

29. A process according to claim 27 or 28, characterized in that the substituted pyridine is aselected from picolines, lutinines and ethylpyridines. o" 4 30. A medicament for the synergistic exertion of 44 a influence of a cancer controlling compound and/or for the reduction of the toxicity of a cancer controlling compound and/or for the protection of human and/or animal cells, characterized in that it contains as the active compound at least one salt according to any one of claims 1 to 6.

31. A medicament according to claim characterized in that the active compound is present in an amount from Img to 500mg per dosis unit. 911028,dbdaL89,59059.res,37 -38-

32. A medicament according to claim 31, characterized in that the active compound is present in an amount of from 5mg to 150mg per dosis unit.

33. A medicament according to any one of claims to 32, characterized in that the medicament is in the form of a parenteral and/or oral pharmaceutical preparation.

34. A method for the synergistic exertion of influence of a cancer controlling compound and/or for the reduction of the toxicity of a cancer controlling compound and/or for the protection of human and/or animal cells which comprises administering to a human or animal in need thereof an effective amount of the medicament according to any one of claims 30 to 33. Compounds of the formula Ia or Ib, processes for their preparation, medicament, containing them or methods involving them, substantially as hereinbefcre described with reference to the Examples. ij DATED this 28th day of October, 1991 Sapec S.A. fine chemicals By Its Patent Attorneys DAVIES COLLISON 911028,dbda89,S9059.res,38