CH638175A5 - N-EPSILON TRIMETHYLLYSINE SALTS, METHOD FOR THE PRODUCTION THEREOF AND MEDICINAL PRODUCTS CONTAINING THESE SALTS. - Google Patents

Description

The invention relates to new N-s-trimethyllysine salts, a process for their preparation and medicaments which contain these salts as active ingredient. The new compounds are the salts formed by N-s-trimethyl-lysine with one or two molecules of aspartic acid, fumaric acid or glutamic acid.

N-e-trimethyllysine has been found in numerous species - from the Actinomycetes to mammals - and in various plants in both bound and free states. The biological significance of this amino acid belonging to the betaine type, as well as that of the related compounds, for example N-dimethyl-lysine, methylated arginine etc., has, however, to date not been able to be clarified exactly [Tyihâk et al .: Life Science 20, 385 (1977)]. For example, N-e-trimethyllysine has recently been isolated from human placenta [T. Tomita and K. Nakamura: Z. Physiol. Chem. 358,413 (1977)], which indicates a physiological or biochemical meaning in the developing organs. The N-s-trimethyl-lysine reacts alkaline in the form of the base and thereby damages the cells. Since it is only bound by receptors in the form of its salts, it is ineffective in vitro. The N-e-trimethyllysine was produced both in itself and for various comparative experiments in the form of its different salts. One of the best studied salts is the dioxalate [T. Takemoto et al .: Yakugaku Zasshi 84, 1176 (1964); Japanese Patent Nos. 24 790 ('65), 09 529 ('68) and 28 092 ('68), French Patent No. 1 442 318, M. Takehara et al .: Nippon Kagaku Zasshi 1969, 101], which is a stable one Salt is, but is out of the question for pharmaceutical use. Among the more thoroughly studied salts are mono- and dihydrochloride [British Patent No. 865,157, French Patent No. 1,442,318, J. Puskâs and E. Tyihâk: Periodica Polytechnica 13, 261 (1969); J.H. Scoly and N.L. Benoiton: Cand J. Biochem. 48, 122 (1970); R.A. Cox and C.L. Hoppel: Biochem. J. 136,1083 (1973)]. However, these salts are very hygroscopic, which makes their storage and metering problematic. Of the salts of N-e-trimethyllysine, the hydrobromide (French Patent No. 1,442,318), the citrate [H. Ozawa et al .: Yakugaku Zasshi 87,935 (1967)], the di-p-hydroxyazo-benzene-p'-sulfonate [R.T. Markiw: Biochem. Med. 13.23

(1975); Y. Kakimoto et al. S. Akazawa: J. Biol. Chem. 245, 5751 (1970); T. Tomita and K. Nakamura: Z. Physiol.

Chem. 358,413 (1977)], the nicotinate (French Patent No. 1,442,318) and the N-acotyl-L-amino succinate (French Patent No. 1,442,318). However, all of these salts do not meet the requirements for stability and pharmaceutical usability.

The aim of the invention is to provide new, pharmaceutically usable N-e-trimethyl-L-lysine salts which are stable, i.e. are not hygroscopic, can be dosed and stored well, have a precisely defined composition and have a pH in aqueous solution that is the same or similar to the pH of the physiological saline solution.

It has now been found that the salts formed by N-e-trimethyl-L-lysine with one or two molecules of fumaric acid, aspartic acid or glutamic acid meet these requirements. N-e-Tri-methylL-lysine monoglutamate (hereinafter TML-Glu) is particularly preferred.

The salts mentioned are prepared according to the invention by reacting N-e-trimethyl-L-lysine in aqueous solution with aspartic acid, fumaric acid or glutamic acid.

The aqueous solutions of the starting materials are expediently combined with one another, the reaction mixture is usually evaporated after the reaction has ended, the residue is recrystallized if necessary and then dried.

The amount of aspartic acid, fumaric acid or glutamic acid used as the starting material naturally depends on whether a salt formed with one or with two molecules of acid is to be produced.

The pharmacological action of the salts according to the invention is illustrated by the following experiments:

The effect of TML-Glu on the blood count of mice infected with NK / Ly tumor was examined.

50 mice of the CFLP O strain (weight: 20-22 g, age: 8 weeks) were injected i.p. infected with NK / Ly ascite tumor (5 x 105 cells per animal). 25 of the 50 animals served as controls, the remaining 25 were on the 2nd, 5th, 9th, 11th, 13th and 19th day after infection (on the 19th day only the 5 surviving animals). ip treated with 100 mg / kg TML-Glu. The blood count was examined on the 2nd, 5th, 8th, 10th, 12th, 13th, 14th and 20th day after the infection. The results are summarized in the following table:

Day of red blood cells white blood cells

Blood formation (millions / mm3) (1000 / mm3)

investigated

Control treated

control

2nd

8.66

7.06

6,446

7,800

5.

7.2

6.95

8,732

8,466

8th.

8.2

7.8

7,200

7,733

10th

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

20th

5.0

2.1

7,133

5,100

It can be seen from the data that the TML-Glu significantly reduces the severe anemia that develops as the NK / Ly tumor grows. The LD50 value of the compound, measured on mice i.p., is above 1500 mg / kg.

Furthermore, the incorporation of thymidine (3H TdR) into the thymus, bone marrow, spleen and mucosa of the small intestine was observed in healthy, 12-week-old female

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Mice of the DBA / 2 strain and NK / Ly ascite tumor cells proliferating in vivo were examined. 5 animals were used per group and time. The experiments were carried out in four repetitions, the TML-Glu was i.p. applied in a dose of 100 mg / kg. It was found that TML-Glu increases the incorporation of 3H TdR in the thymus by 85 + 7%, in the spleen by 75 + 8% and in the NK / Ly tumor cells by 94 + 11% (24 hours after treatment). The cells of the bone marrow showed an increased (57 + 4% and 72 + 5%) incorporation of 3H TdR in the 10th and 48th hour. In the NK / Ly tumor cells, the incorporation increased even more in the 48th hour (124 + 14%). At i.p. Application, the incorporation of 3H TdR into the mucosa of the small intestine was not increased, whereas in the case of administration p.o. A 32 + 9% increase in paving was observed 32 hours after treatment. This shows that the cells have started DMS synthesis and have entered the active phase.

The transforming effect exerted on the lymphocytes was also investigated in human cultures. Lymphocytes were isolated from the peripheral blood of healthy blood donors using the Ficoll-Uromiro gradient. These were suspended in a number of 5 × 105 / ml cells in Parker 199 medium containing autologous serum and incubated at 37 ° C. for 96 hours. Some of the samples were treated with 100 | xg / ml TML.2HC1, another part with 100 ng / ml TML-Glu, the remaining samples served as controls. The transforming effect was determined by the degree of incorporation of 3H TdR, the treatment with 3H TdR was carried out in a dose of 11 jiCi / ml 3 hours before the incubation was stopped. Culturing was discontinued 48, 72 and 96 hours after the start. The activity bound to DNA was determined after extraction with boiling perchloric acid by liquid scintillation. The following results were obtained:

Time (h) recording of 3H TdR (cpm)

TML-2HC1

TML-Glu

control

48

146

594

123

-

756

167

413

72

295

3010

159

303

2427

254

1413

96

231

2825

271

330

2878

301

3659

The fact that the resting cells got into the active phase proves that they started to multiply. For this reason, the development of the total number of cells in the tumor was also examined.

20 female mice of the CFLP strain were inoculated with NK / Ly tumor. 24 hours later, 10 animals were administered 100 mg / kg TML-Glu daily for 8 days, while the remaining 10 animals served as controls. The mice were sacrificed 9 days after vaccination, the weight of the animals and the total number of cells of the tumor were determined. It was found that the total number of cells in the treated animals was 35% higher than in the control.

Finally, the effect of TML-Glu on the humoral and cellular immune response in mice was examined. Levamisole (L-6-phenyl-2,3,5,6-tetrahydroimidazo [2, l-b] thiazole) was used as the reference substance. The investigations were carried out on groups of 10 female, 2-month-old mice of the DBA / 2 strain, the red blood cells of the sheep i.p. used. E. coli endoxin and a single treatment with 2-5 mg / kg levamisole were given as a positive control. The TML-Glu was dosed at 5 x 100 mg / kg i.p. for two weeks. as pretreatment or as a single treatment with 100 mg / kg i.p. also applied before the administration of the antigen. The haemagglutinin titer was determined using the Takâcs microtitrator. It was found that the one-time treatment with TML-Glu was ineffective, but the repeated pretreatment caused an increase in titer as great as that of the levamisole. However, this has various side effects, for example it has a harmful effect on the gastrointestinal tract. TML-Glu, on the other hand, has no side effects, it is the salt of a substance that also occurs in the organism.

To investigate the cellular immune response, Legrange et al. described experimental model applied. Its essence is that after repeated antigen administration (red blood cells of the sheep) the sole thickness of the mice increases. The extent of this growth is generally reduced by the agents that stimulate the humoral immune response based on the control, but is increased after a few days compared to this already decreased control value; this also occurs with pretreatment with TML-Glu (5 x 100 mg / kg, within two weeks).

Based on the test results described, the advantages of TML-Glu can be summarized as follows:

1. Since it makes numerous cells sensitive to cytostatics, it can be used in combination with cytostatics in the treatment of tumors.

2. As a result of its effect on the blood picture and the effect on the intestinal mucosa which increases the proliferation, the compound applied at a suitable point in time reduces the toxic effect of the cytostatics.

3. When administered alone, the compound improves the blood picture in the case of tumor anemia, but also in the case of anemia of other origin.

4. The immunostimulating effect of the compound can be used well in tumor diseases, but also in other cases associated with a reduction in humoral immunity.

The invention is explained in more detail with reference to exemplary embodiments. The abbreviations used in the examples correspond to the abbreviations common in the specialist literature [J. Biol. Chem. 247, 977 (1972)]. The abbreviation TML stands for N-e-Trimethyl-L-lysine.

A Rotavapor apparatus (Büchi) was always used to evaporate the new compounds. The melting point was determined using the Tottoli (Büchi) apparatus. The thin layer chromatograms were recorded on fixion layers. The solution of 50 g of citric acid, 50 g of NaOH and 7 ml of concentrated hydrochloric acid in 500 ml of distilled water was used as the mobile phase. The chromatograms were developed with a solution of 80 ml of methanol, 20 ml of glacial acetic acid, 0.2 g of ninhydrin and 0.1 g of copper (II) sulfate.

example 1

N-e-trimethyl-L-lysine monoglutamate

50.0 g (0.265 mol) of chromatographically uniform N-trimethyl-L-lysine are dissolved in 500 ml of distilled water. The solution is heated to 50 ° C and with the Lö5

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solution of 39.0 g (0.265 mol) of L-glutamic acid combined in 500 ml of distilled water. The solution obtained is stirred at 50 ° C. for 30 minutes and then 5 g of Norit activated carbon are added. The solution is stirred for a few minutes, then filtered and evaporated to dryness under reduced pressure. When evaporating, the temperature of the solution must not rise above 50 ° C. The crystalline residue obtained is dried in vacuo over phosphorus pentoxide to constant weight. 95-98 g of N-s-trimethyl-L-ly-sin-monoglutamate are obtained, which melts at 104-107 ° C. [a] 2j§ = + 5.15 ° (c = l, water).

Amino acid analysis: Glu: 1.0 (1); TML: 1.13 (1). Elemental analysis for C14.H30O6N3 • 2H20:

calc .: C 45.2% N 11.3%

Found: C 45.6% N 11.2%.

Example 2 N-s-trimethyl-L-lysine difumarate 7.0 g (0.37 mol) of chromatographically uniform N-e-trimethyl-L-lysine are dissolved in 70 ml of distilled water. The boiling solution of 99.5 g (0.85 mol) of fumaric acid in 4000 ml of ethanol is added to the solution with stirring. The slowly cooled mixture is held at 0 QC for 48 hours. The precipitated crystals are filtered off, washed first with ethanol, then with ether and finally dried. 108 g (69%) N-s-trimethyl-L-lysine-5 difumarate are obtained. This is recrystallized from ethanol containing 3% water. The product melts with decomposition at 147-149 ° C. Rf: 0.1; [apfj = + 6.0 ° (c = 2, water). Amino acid analysis: TML: 1.0 (1).

Example 3

N-s-trimethyl-L-lysine diaspartate 50.0 g (0.265 mol) of chromatographically uniform N-s-trimethyl-L-lysine are dissolved in 500 ml of distilled water at 50 ° C. This solution is added to a 50 ° C 15 warm solution of 70.1 g (0.53 mol) of L-aspartic acid in 500 ml of distilled water. The solution is stirred for a few minutes and then 5 g of Norit activated carbon are added. After filtering, the solution is evaporated under reduced pressure. The oily residue is solidified by treating it with dry ethanol. 60.0 g of N-e-trimethyl-L-lysine diaspartate are obtained, which melts at 142-153 ° C. (The substance is very hygroscopic).

Claims (6)

638 175 1. The salts formed by N-s-trimethyl-L-lysine with one or two molecules of fumaric acid, aspartic acid or glutamic acid. 2. N-s-trimethyl-L-lysine monoglutamate according to claim I. 2nd PATENT CLAIMS 3. N-s-trimethyl-L-lysine difumarate according to claim 1. 4. N-e-trimethyl-L-lysine diaspartate according to claim 1. 5. A process for the preparation of salts of ne-tri-methyl-L-lysine with one or two molecules of fumaric acid, aspartic acid or glutamic acid, characterized in that Ns-trimethyl-L-lysine in aqueous solution with a corresponding amount of fumaric acid, Aspartic acid or glutamic acid. 6. Pharmaceutical preparations, characterized in that they contain as active ingredient at least one salt of N-s-trimethyl-L-lysine formed with one or two molecules of fumaric acid, aspartic acid or glutamic acid.