CA1113958A

CA1113958A - N-.epsilon.-TRIMETHYL-L-LYSINE SALTS

Info

Publication number: CA1113958A
Application number: CA313,224A
Authority: CA
Inventors: Karoly Lapis; Bela Szende; Andras Jeney; Zsuzsa Suba; Laszlo Kovacs; Erno Tyihak; Lajos Kisfaludy; Jajos Kovacs; Gyorgy Dobo; Bela Szarvady
Original assignee: Richter Gedeon Vegyeszeti Gyar RT
Current assignee: Richter Gedeon Vegyeszeti Gyar Nyrt
Priority date: 1977-10-13
Filing date: 1978-10-12
Publication date: 1981-12-08
Also published as: FR2405925A1; AU520254B2; FR2405925B1; DE2844501A1; DK148355C; YU239378A; NL7810331A; CH638175A5; ATA714578A; DK148355B; IL55645A0; BE871196A; DK455378A; JPS5858340B2; YU40987B; IL55645A; JPS5463014A; AT360501B; GB2006207B; HU174503B

Abstract

ABSTRACT OF THE DISCLOSURE

There are disclosed new N-?-trimethyl-L-lysine salts, a process for the preparation of same and pharmaceutlcal compositions containing them. More particularly, novel salts of N-?-trimethyl-L-lysine, formed with one or two molecules of aspartic acid, fumaric acid or glutaminic acid are described. The new salts are stable and may be advantageously used for different pharmacological purposes.

Description

This invention relates to novel N--trimethyl-L-lysine salts, a process for the preparation thereof and pharmaceutical compositions contain-ing the sa~e.
More particularly, this invention relates to novel salts of N--trimethyl-L-lysine formed with one or two molecules of aspartic acid, fumaric acid or glutamunic acid.
N-~-trimethyl-L-lysine is a kncwn compound which has been detected in numerous species including Actinomycetes and mammals as well as in a lot of different plants either in a bound or in a free form. The exact bio-logical role of this compound, which belongs to the basic amino acids of the betaine-type, and of the closely related compounds~ such as N-dimethyllysine, methylated arginines etc., however, has not been revealed up to the present time [Tyihak et al.: Life Science, 20 (1977) 385]. The fact that N-~-tri-methyl-L-lysine has been lately isolated from human placenta [T. Tomita and K. Nakamura: Z. Physiol. Chem. 358 (1977) 413] may be indicative of its physiological or biochemical role in the growing organisms.
The free N--tximethyl-L-lysme base is injurious to the cells and, since it can be attached to a receptor only in the form of a salt, it is in-active in in vitro tests. Due to the above facts N--trLmethyl-Lrlysine has always been isolated and used for various investigations in the form of its salts. Gne of the salts which have been the most widely investigated is the dioxalate [T. Takemoto et al., Yakugaku Zasshi 84 (1964) 1176]; Japanese Patent Sp~cifications No. 24,790, (1965), 09,529 (1968) and 28,092 (1968), French Patent Specification No. 1,442,318 and M. Takeh æ a et alO: Nippon Kagaku Zasshi 101 (1969)], which is stable but obviously cannot be used for pharmacological purposes. Other much-examined salts are the corresponding mcno- and dihydrochlorides [British Patent Specification No. 865,157; French Patent Specification No. 1,442,318; J. Puskas and E. Tyihak: Periodica Poly-technica 13, (1969) 261; J. H. Seely and N. L. Benoitan, Cand. J. Biochem. 48 (1970) 1122; R. A. Cox and C. L. Hoppel, Biochem. J., 136 (1973) 1083].

~k ' These salts are however, very hygroscopic, what is highly disadvantageous with respect to their storability and dosage. Cther salts of N-~-trimethyl-L-lysine, for instance the hydrobromide [French Patent Specification No.
1,442,318], citrate [H. Ozawa et al., Yakugaku Zasshi 87 (1967) 935], di-p-hydroxyazobenzene-p'-sulphonate [R. T. Markiw: Biochem. Med. 13 (1975) 23:
Y. Kakimoto and S. Akazawa., J. siol. Chem. 245 (1970) 5751; T. TQmita and K. Nakamura, Z. Physiol. Chem. 358 (1977) 413], nicotinate [French Patent Specification No. 1,442,318] and N-acetyl-L-amino-succinate thereof [French Patent Specification No. 1,442,318] have also been described, but these salts do not ccmply with the requirements of stability and practical application.
The object of the present invention is to provide new, pharmaceutic-ally applicable N-~-trimethyl-L-lysine salts, which are stable, i.e. are not hygroscopicJ have a good storability, are easy to convert into dosage forms, have a definite composition and in aqueous solution have a pH-val~e approxi-mately identical with that of the physiological sodium chloride solution.
It has now been found tllat the salts of N-~-trimethyl-L-lysine formed with one or two molecules of fumaric acid, aspartic acid or glutaminic acid fulfil the a~ove requirements. The N--trimethyl-Lrlysine monoglutamate ~furtheron designated as TML. Glu) proved to be especially advantageous.
Acoording to a further aspect of this invention the above salts are prepared by reacting N--trimethyl-L-lysine with aspartic acid, fumaric acid or glutamunic acid in an aqueous solution.
According to a preferred embodlment of the process the aqueous solu-tion5 of the starting materials are combined, and, when the reaction termin-ates, the reaction mlxture obtained is evaForated and the evaporation residue - if desired æ ter recrystallization - is dried.
The quantity of the aspartic acid, fumaric acid and glutaminic acid, used as a starting material, depends on the product to be prepared, i.e. on that if the salt to be prepared is formed with one or two molecules of a corresponding acid.

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The phanmacological properties of ~he cnmpounds according to the invention are illustrated by the follcwing tests.
me influence of IML.Glu on mice, into which NK/I.y tumour cells were transplanted, was investigated. me tests were performed as follows:
Into 50. eight-week old CFLP o mice, weighing 20 to 22g each NK/Ly ascites tumour cells were transplanted intraperitoneally, in a dose of 5x105 cells/
anLmal. 25 animals were used as a control and the re ~ining 25 am mals were administered 100 mg./kg. i.p. doses of TML.Glu on the 2nd, 5th, 9th, 11th, 13th and l9th day follcwing the transplantation of tumour cells. On the l9th day only the 5 animals which were still alive were given the above treatn~lt.
me effect of the treatment on the blood count of the animals is illustrated in the ollowing table.

Day of determination Red blood cellsWhite blood cells of the blood count ~millions/mm3)(thousands/mm.3) following the transplantation treated control treatedcontrol _ _ .

2. 8.66 7.06 6.466 7.800 5. 7.2 6.95 8.732 8.466 8. 8.2 7.8 7.2Q0 7.733 10. 7.2 6.4 8.520 8.900 12. 7.5 5.7 10.800 6.500 13. 6.6 4.9 8.260 6.700 14. 5.5 4.5 9.000 7.700 20. 5.0 2.1 7.133 5.100 ~ . _ . . _ . _ _ _ The above data unambiguously show tha~ the serious anaemia due to the growth of NK/Ly tumour is considerably reduced when the animals are treated with TML.Glu. LD50 i.p. on mice = over 1500 mg./kg.
Tests were carried out to investigate the distribution of thymidine (3H TdR) into the thymus, bone marrow, spleen an ileum mucous m~nbrane of healthy, 12 weeks old DBA/2 female mice and into in vivo proliferating NK/Ly ascites tumQur oells. Tests were performed on groups consisting of five animals and were repeated four subsequent times. TML.Glu was administered in an i.p. dose of 100 m~./kg~ It has been found that the incorporation of the H TdR into the thymus was increased by 85 +7 %~ into the spleen by 75 +8 %
and into the NK/Ly tumour cells by 94 +11 % in the 24th hour following the treatment with the abo~e dose of TML.Glu~ In the bone marrow cells in the 10th hour the incorporation of 3H TdR was increased by 57 +4 ~ and in the 48th hour by 72 +5 %. In the case of NK/Ly tumour cells the incorporation was further increased even in the 48 hour when it amounted to 124 _14 %.
When TML.Glu was administered intraperitoneally the incorporation into the ileum mucous membrane was not increased, but in the case of oral admim stra-tion a 50 _9 % increase was observed in the 32nd hour following the treatment.
The increase in the incorporation of 3H TdR indicates that the cells started synthetizing DNS, i.e. turned active.
The transfon~ng effect exerted by the new N-~-trimethyl-L-cell lysine salts on lymphocyta cells was tested also on human cultures. From the peripheral blood of healthy donors, lymphocytes were isolated by means of the Ficoll-Uromiro gradient. m e lymphocytes were suspended in a Parker 199 medium containing an autologe serum at 20 &, in a concentration of 5x105 oe lls/ml. and subsequently were incubated at a temperature of 37 C for 96 hours. The cultures were divided into three groups. The first group was treated with a 100 ~g/ml. dose of TML.2HC1, the second one with a 100 ~g/ml.
dose of TML.Glu while the third group served as a control. The transforming effect was characterized by the incorporation of H TdR. The H TdR was administered in a concentration of 11 ~Ci/ml., -three hours before the inter-ruption of cultivation. The cultivation was interrupted 48, 72 and 96 hours after the start. The activity bound to DNS was determined after extraction with hot perchloric acid, by a liquid scintillation technique. T~e res~ts obtained æe set forth in the following table.

Tlme 3H TdR incorporation (hours) TML.2HCl TML.Glu Cbntrol -_ 756 330 2878 ' . .
The assumption that the cells turn active, among others, is proved by the fact that they start multiplying. The shaping of the total num~er of tumour cells was, therefore, also determined.
Into about 20 female CF$P mices ~K/Ly tumour cells ~ere trans-planted. 10 animals were administered a 100 mg./kg. daily dose of TML.Glu for 8 subsequent days starting 24 hours after the transplantation of tumour cells. 10 mices were used for comparison. Mice were killed 9 days after treatment, and the weight of the animals and the total number of tum3ur cells were determlned. It was found that the treatment resulted in a 35 % increase of the total nu~ber of tumour cells related to the control.
Finally the influence of TML.Glu on the humoral and cellular immune response of mioe was observed. For oomparison L-6-phenyl-2,3,5,6~tetra-hydroimidazo[2,1-b]-thiazole (Levamisol) was used~ Tests were performed on groups consisting of 10 two-week old female DBA/2 mice each. As an antigen, red blood cells of sheeps were used, which were administered intraperitone-ally. As a positive control, E. coli endotoxin and a single 2 to 5 m~./kg. of body weight dose of Lovamisol were administered. Before the ~dministration of the antigen, with 2 weeks five 100 mg./kg. of bcdy weight i.p. and one 100 ,~, ~3 .

m~g./kg. of body weight i.p. doses of TMLoG1u were administered. The haemagglutinine titre was determined by a "Takacsy" microtitrator. It was found that a single pretreatment with TML.Glu had no observable effect, but a repeated pretreatment gave about the same increase in the titre-value as Leva~isol. sut while Levamisol has various undesired side-effects, for exa~ple it is injurious to the gastro- m testinal tract, TML.Glu is devoid of such side-effect, since it is the salt of a natural substance, which is pre-sen in the living organism.
The cellular immune response was investigated on the basis of the experimental m~del described by Legrange et al. This model is based on the observation that after a repeated addition of red blood cells (sheeps) the paw~thickness of mice is increased. Generally speaking, those agents, which stimulate the humoral i~mune response, initially decrease the grcwth - in ccn~rision with the control - thenr after several days, produce an increased grcwth, related to the control, in whi~h the growth in the meantime has slowed dcwn. me same trend was observable, in about tw~ weeks, when mioe were pretreated with five 100 mg./kg. of bcdy weight doses of IML.Glu.
On the basis of the results obtained in the tests described above, TML.Glu has the following main advantages:
1) Sin oe it sensibilizates numerous oe lls against cytostatics, it can be advantageously used in the chemotherapy of tumorous diseases in combina-tion with knGwn cytostatic agents.
2) Sin oe it has a prolipheration increasing effect on the blood count and on the ileum mucous NEmbrane, it decreases the toxid ty of cytostatics, when is used in an appLnpriate time.

3) it has a keneficial influence on the blood count, even when admin-istered alone, and therefore can successfully be used in the treatment of tumorous anaemia and anaemias of different origin.

4) Its immune-stimulating effect can be utilized in the treatment of tum~rous and other diseases, which are accompanied by the decrease of -the humoral immunity.

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The other salts according to the inve~tion possess about the same advantages.
The process according to the invention ic~ further illustrated by the following, non-limiting Examples. The abbreviations u~sed in the Examples and throughout the specification are these generally used in the literature [J. Biol. Chem. 247, (1972) 977]. The most frequently used abbreviation, TML = N-E-trimethyl-L-lysine.
Duling the preparation of the compounds according to this invention evaporation is always accomplished in a BUChi Rotavapor equipment. The mel-t ing points were determined in a Tottoli (Buchi) apparatus. Thin layer chromatography was effected on a Fixlon layer. As a solvent mixture, a mix-ture of 50 g. citric acid, 50 g. of sodium hydroxide, 7 ml. of a concentrated hydrochloric acid and 500 ml. of distilled water was used. me chrcmatograms were developed with a n~Lxture of 80 ml. of methanol, 20 ml. of glacial acetic acid, 0.2 g. of ninhydrine and 0.1 g. of cupric sulphate.
Example 1 N-~-trimethyl-L-lysine monogluta~ate 50.0 g. (0.265 moles) of chrcmatographically unifo~n N-c-trLmethyl-L-lysine are dissolved in 500 ml. of distilled water. The mixture is heated up to 50 C and is ccmbined with a solution of 39.0 g~ (0.265 moles) of L-glutaminic acid in 500 ml. of distilled water, having the same temperature.
me solution obtained is stirred at 50 C for 30 minutes, then is decoloured with 5 g. of Morit charcoal. After several minutes' stirring the solution is filtered and evaporated to dryness under reduced pressure. Care must be taken that the temperature of the solution should not exceed 50 CO The crystalline evaporation residue is dried in vacuo, in the presence of phos-phorous pentoxide, up to a steady weight.
Yield: 95 to 98 g. of N-~-trimethyl-L,lysine monoglutamate; melt-ing point: 104 to 107 C;
[]25 = +5.15 (c = 1, water)O

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Pmdno acid analysis Glu: l.G(l): TML: 1.13(1).
AnalysiS for Cl4H30o6N3 2 Calculated: c = 45.2 ~, N = 11.3 ~;
Found: C = 45.6 %, N = 11.2 %.
Example 2 N--trimethyl-L-lysine difumarate 7.0 g. (0.37 moles) of chromatographically uniform N-~trimethyl-L-lysine are dissolved in 70 ml. of distilled water. To ~he solution obtained a hot solution of 99.5 g. (0.85 moles) of Eumaric acid in 4000 ml. of ethanol is added with stirring. After a slow cooling, the mixture is allcwed to stand at 0 & for two days. me precipitated crystals are filtered off, washed with ethanol and subsequently with ether and dried. 108 g. (69 %) of N--trimethyl-L-lysine difumarate are obtained, which are then recrystallized from ethanol, ~ontaining 3 ~ of water.
Melting point: 147 to 149 & (decomposition~;
Rf = 0.1, [~]D = +6.Q (c = 2~ water).
Amino acid analysis: T~: 1.0(1).
Example 3 N--trimethyl-L-lysine diaspartate .
50.0 g. (0.265 moles) of chromatographically uniform N--trimethyl-L-lysine are dissolved in 500 ml. of distilled water, having a temper~ture of 50 C. The solution obtained is added into a solution of 70.1 g. (0.53 mole~) of L-aspartic acid in 500 ml. of distilled water, which has the same tempera-ture. After some minutes' stirring the solution is decoloured with 5 g. of Norit charcoal, the charcoal is filtered off and -the filtrate is evaporated under reduced pressure. The remaining oily substance is treated with dry ethanol to afford 60.0 g. of solid N--trimethyl-L-lysine diaspartate.
Melting point: 142 to 153 C (the substance is very hygroscopic~.

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Claims

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

1. A process for preparing a salt of N-.epsilon.-trimethyl-L-lysine with fumaric acid, aspartic acid or glutaminic acid, which process comprises re-acting N-.epsilon.-trimethyl-L-lysine with fumaric, aspartic or glutaminic acid in aqueous solution.

2. A process according to claim 1 wherein the N-.epsilon.-trimethyl-L-lysine is reacted with one molecule of acid to form the monofumarate, monoxaspar-tate or monoglutamate salt.

3. A process according to claim 1 wherein the N-.epsilon.-trimethyl-L-lysine is reacted with two molecules to form the difumarate, diaspartate or di-glutamate salt.

4. A fumarate, aspartate or glutaminate salt of N-.epsilon.-trimethyl-L-lysine when prepared by a process according to claim 1 or an obvious chemical equivalent thereof.

5. A process for preparing N-.epsilon.-trimethyl-L-lysine monoglutamate which comprises reacting N-.epsilon.-trimethyl-L-lysine in aqueous solution with an equimolar amount of glutamic acid in aqueous solution.

6. The compound N-.epsilon.-trimethyl-L-lysine monoglutamate when prepared by a process according to claim 5 or an obvious chemical equivalent thereof.

7. A process for preparing N-.epsilon.-trimethyl-L-lysine difumarate which comprises reacting N-.epsilon.-trimethyl-L-lysine in aqueous solution with a bimolar amount of fumaric acid in ethanol.

8. The compound N-.epsilon.-trimethyl-L-lysine difumarate when prepared by a process according to claim 7 or an obvious chemical equivalent thereof.

9. A process for preparing N-.epsilon.-trimethyl-L-lysine diaspartate which comprises reacting N-.epsilon.-trimethyl-L-lysine in aqueous solution with a bimolar amount of aspartic acid in aqueous solution.

10. The compound N-.epsilon.-trimethyl-L-lysine diaspartate when prepared by a process according to claim 9 or an obvious chemical equivalent thereof.