DK155433B - METHOD OF ANALOGUE FOR THE PREPARATION OF AMINO ACID DERIVATIVES OR PHYSIOLOGICALLY ACCEPTABLE SALTS THEREOF - Google Patents

Description

DK 155433 B

The present invention relates to an analogous process for the preparation of novel amino acid derivatives having valuable biological and pharmaceutical effects.

The amino acid derivatives prepared by the process of the invention have the general formula 5

NH - CH-COOH

2 I

Wn 1 CO-N-CH- (CH,), - B1, 10, R 2 where B is a group of the formula -SC 2 OH or -ΟΡΟ (ΟΗ) 2 / R is a hydrogen atom or an alkyl group of 1- 4 carbon ** 15 atoms, 2 R is a hydrogen atom or a carboxyl group, n is 1 or 2, and t is 1 or 2, or are physiologically acceptable salts or optically active antipodes thereof.

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These compounds have valuable biological or pharmacological effects. All the compounds of the invention are novel.

Among the compounds of the invention, due to its biological effect, v-L-25 glutamyltaurine corresponding to the formula h.n-ch-cooh 2 in CKp

30 [* XXIV

CHO I 2 CO-NH-CH2-CH2-SO20H and which has a broad therapeutic and preventive spectrum of action against morbid changes that can be traced back to damage by "AGAS" (the aerobiospheric genetic adaptation system).

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DK 155433 B

To illustrate the concept of "AGAS", the following are the main tissues and organs that form this system.

(a) all biological interfaces in contact with the external air such as the biosphere (skin and skin formation, eye cornea 5 and Conjunctiva, oral and pharyngeal cavities, respiratory tract and lung); b) skeleton and joints (tubular and spongy bones, joints, synovial membranes * skeletal muscle); (c) the organs involved in the regulation of the ion household (transepithelial transport system: intestinal tracts and kidney channels); (d) the tooth decomposition of the nourishment required by the tooth (in tandave) clay with roots); e) hearing, smell and voice organs.

Thus, the compounds of this invention exert a favorable biological or therapeutic effect on the organs or tissues of the AGAS system enumerated herein.

In addition, the compounds of the present invention act upon the following functions associated with the AGAS system: radiation protection, favoring wound healing, common activating effect on mesenchymal, protection against the ever-growing infection and contamination risk of skin and mucosa ( the lysozyme production of the moist mucosa, activation of the gut epithelium in the respiratory tract, etc.), increased protection against the infections caused by viruses and fungi.

The compounds of the present invention are effective against the ever-increasing stress effects associated with mainland life (e.g., meteorological influence, large differences between day and night temperature, increased risk of injury) as they stabilize. adaptation syndrome and at the same time mitigates the peripheral tissue damage of the glucocorticoids (such as damage to the connective tissue, injury to bone matrix components, etc.). Development of immune homeostasis (increasing body recognition of which cells are capable and which are not).

The compounds prepared by the process according to the invention exert their effect partly immediately, 3

DK 155433 B

partly through the regulation of vitamin A metabolism, by the production of vitamin A metabolites with a stronger polar character.

This effect can be compared to the effect of the parathormone on the 25-hydroxycholecalciferol-1α-hydroxylase enzyme in the renal tract. The directions of action of the compounds are as follows: 5 A) Vitamin A grade effects: a) Increasing effect of pharmacological and biochemical effects on chondroitin sulfate synthesis; beneficial effect on the wound healing or on the experimentally reduced wound healing by cortisone in rats and dogs; potentiating effect on vitamin A's effect in rats and chickens in experimentally induced hypo- or hypervitaminosis; attenuating effects on the ulcer ation-related stress effects in rats; beneficial effect on the degranulation of mastocytes; increasing effect on the production of lysozyme; effect on household traceability (silicon, zinc, copper, manganese, fluorine); promoting effect on epithelial formation; promoting effect on the alkaline phosphatase activity; the effect on the granuloma bag formation (Granulomsackbildung) caused by local action of vitamin A; the extremely flat course of the dose-effect curve or the change of effect sign at large doses; activating effect on the Golgi apparatus; beneficial effect on the formation of mucus or goblet cells; increasing effect on the concentration of vitamin A.

B) Clinical therapeutic effects: keratoconjunctivis sicca; Sjogren's syndrome; rhino-laryngo-pharingitis sicca; ozæna; chronic bronchitis; sinobronchitis; mucoviscidosis; constitutional lung disease in young children; periodontal disease; the susceptibility of skin and mucous membranes to viruses and fungi; cortisone antagonistic effect; beneficial effect on healing of operative and mucosal wounds; erosio colli; pruritus-like disorders; reducing the sense of smell and taste.

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DK 155433 B

4 B) Effects - other than Vitamin A grade a) Pharmacoligial and biochemical effects: effect on blood sugar levels with regard to a transient reduction; increasing effect on phosphaturia, lowering effect on phosphate level 5 in serum; radiation protective effect; reducing the time required to reach the goal of maze trials in inactivated animals; diminishing effect on experimentally induced fluorine and cadmium toxicosis; increasing effect on the cyclic adenosine monophosphate depletion of the kidneys; attenuating effect on the symptoms of experimentally induced lathyrism; decrease in histamine sensitivity; enhancing effect on liver enzyme tyrosine aminotransferase activity.

b) Therapeutic effects: weak radiation damage; 15 vitiligo; muscle hypotonia; psychoenergetic effect; beneficial effect on involutional and gerontological conditions as well as on the mnestic functions; keloid inclinations; spondylosis ankylo poetica; diseases of the movement organs due to wear and tear; scle-rotic fundus; amyloidosis; morphea; fibrocytic mastopathy.

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The compounds of the process according to the invention can be used for addition to drug mixtures. They improve vitamin A metabolism. In addition, the resorption of trace elements by the compounds is increased as the concentration of trace elements in the blood is increased.

In the veterinary medicine, the compounds prepared according to the invention have similar fields of application as in human medicine, ie. eg skin damage (peeling), wound healing and bone fractures.

The process according to the invention is characterized by the characterizing part of the claim.

The process according to the invention is illustrated in the following by means of some examples.

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DK 155433 B

ISLAND

Example 1 a) Carbobenzyloxy-γ- (g-benzyl) -L-glutamyltaurine 40.85 g (0.11 mol) of carbobenzyloxy-L-glutamic acid α-benzyl ester (Liebigs Annalen 655, 200, 1962) are dissolved in 500 ml of acetonitrile. The solution was cooled to -15 ° C to exclude the humidity. To the solution is added dropwise with stirring, first, 15.4 ml (0.11 mole) of triethylamine and then 15.4 ml (0.11 mole) of chloromyric acid isobutyl ester. The reaction mixture is stirred at -15 ° C for 40 minutes and then 28 ml (0.2 mole) of triethylamine is added followed by 11.26 g (0.05 mole) of cystamine hydrochloride and finally 250 ml of acetonitrile. The mixture is stirred at -15 ° C for a further two hours and then for another four hours at room temperature.

After the reaction time expires, the mixture is evaporated at 30 ° C

under vacuum. The residue is taken up with stirring and cooling in 200 ml of ice water and the mixture is evaporated again at 35 ° C under vacuum. The residue, together with 250 ml of water and 500 ml of ethyl acetate 1, is introduced into a separatory funnel and the organic phase is separated. In the said order, the organic phase is first shaken with 250 ml of water, then twice with 250 ml of 5% sodium carbonate solution each time and then twice with 250 ml of 1N hydrochloric acid and finally with 250 ml of water. (From the aqueous phase obtained by shaking with sodium carbonate solution, by acidification with hydrochloric acid and shaking with ether, about 5 g of unreacted carbobenzyloxy-L-glutamic acid and benzyl ester can be recovered). The ethyl acetate phase is dried over anhydrous sodium sulfate and then evaporated under vacuum at 30 ° C to dryness. There is obtained a 25 thick, oily residue which quickly solidifies to a crystalline mass. This is torn with 250 ml of absolute ether and the crystals are filtered off. The crude product (40-42 g) is recrystallized from a mixture of 100 ml of ethyl acetate and 170 ml of ether. 29.3 g of N, N '-bis- / N-lcarbobenzyloxy-γ- (α-benzyl) -L-glutamyl7-cystamine are obtained, mp 91-92 ° C.

Calcd for CMH.NN, 0inSo (M = 859.05): C 61.52 H 5.89 N 6.52 S 7.46 Found: C 60.85 H 5.91 N 6.61 S 7.72% 35 6

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25.77 g (0.3 mole) of the above-obtained N, N'-bis- N-Icarbobenzyloxy-γ- (α-benzyl) -L-glutamyl7-cystamine are dissolved in 75 ml of glacial acetic acid. The ice-cooled solution is added dropwise over 15 minutes to a freshly prepared mixture of 75 ml of 30% hydrogen peroxide and 225 ml of glacial acetic acid. After addition 5, the cooling is removed and the reaction mixture is stirred for four hours at room temperature. Then it is evaporated under vacuum at 30 ° C. The oily product is dried in an electric silencer first over phosphorus pentoxide and then over solid potassium hydroxide. 28.5 g of lcarbo-benzyloxy-γ- (α-benzyl) -L-glutamyltaurine are obtained. The crude product can be used without purification to prepare γ-L-glutamyltaurine.

b) Ύ-L-QLutamyltaurine 5.79 g (12.1 mmol) of the carbobenzyloxy-γ- (α-benzyl) -L-glutamyltaurine prepared in accordance with Example 1 a) are dissolved in a mixture of ethanol (100 ml) and 25 ml. water and hydrogenated with shaking in the presence of 0.5 g of 10% palladium carbon as a catalyst. After the hydrogen consumption has ceased, the solution is filtered and evaporated at 30 ° C under vacuum. The oily residue is dried in desiccator over phosphorus pentoxide. 3.1 g of γ-L-glutamyltaurine is obtained, which is easily dissolved in water but not in alcohol. By adding a small amount of water and alcohol in small portions, the product can be crystallized. The crystalline crude product melts at 202-204 ° C.

The crude product is recrystallized several times from 80% ethanol. 2.02 g of pure final product is obtained as calculated from N, N'-bis- / N-carbobenzyloxy-γ- (α-benzyl) -L-glutamyl7-cystamine corresponding to a yield of 66%. The pure product melts at 219-220 ° C. = + 14 ° (water, c = 1.02). The relative mobility to cysteic acid by paper electrophoresis carried out at pH 6.5 out-of-30 makes 0.73, at pH 1.8 0.53.

Rp (n-Butanol / pyridine / glacial acetic acid / water 15: 10: 3: 12) = 0.19.

Calcd for CyHy 2 O 4 S (m = 254.27): C 33.07 H 5.55 N 11.02 0 37.75 S 12.61 Found: C 33.15 H 5.76 N 10.94 0 37, 53 S, 12.17%.

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DK I 'i54':; 3 B

Example 2 a) )- (α-Benzyl) -L-glutamyltaurine 26.32 g (55 mmol) of the carbobenzyloxy-γ- (oc-benzyl) -L-glutamyltaurine prepared in Example 1 a) are dissolved in 50 ml of ice-cream. 5 acetic acid. To the solution is added 4 moles of hydrocarbon dissolved in 50 ml of glacial acetic acid. Lively carbon dioxide evolution is observed. The reaction mixture is left at room temperature for two hours and then evaporated under vacuum at 30 ° C. Dissolve the oily residue in 170 ml of water and shake five times with 70 ml of ether each time.

The aqueous phase is evaporated at 35 ° C under vacuum. 20.42 g of γ- (α-benzyl) -L-glutamyltaurine are obtained, which is recrystallized from 90% ethanol.

Rp (n-Butanol / pyridine / glacial acetic acid / water 15: 10: 3: 12) = 0.53 Rp (n-butanol / glacial acetic acid / water 4: 1: 1) = 0.39.

B) γ-L-Glutamyltaurine

The γ- (oc-benzyl) -L-glutamyl taurine (20.42 g) obtained in Example 2a) is dissolved in 150 ml of 1 N potassium hydroxide solution. The solution is left for four hours at room temperature and then transferred to a column of 2 x 100 cm filled with "Dowex" 50 x 2 (Fluka, 100-200 mesh) which is in the H cycle. Elute with water. From the beginning of the washout, 300 ml of eluate is collected. This is evaporated under vacuum at 35 ° C. The oily residue is crystallized by the addition of 8-10 ml of water and ca. 100 ml of ethanol.

After filtration, washing with alcohol and drying, 13.7 g of γ-L-glutamyltaurine are obtained. The crystalline product is recrystallized from 80% aqueous ethanol. 9.79 g of pure product are obtained as calculated from N, N'-bis- / N-lcarbobenzyloxy-γ- (a-benzyl) -L-glutamyl7-cystamine corresponding to a yield of 10%.

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Example 3? -L-Glutamyltaurine 5.42 g (11 mmol) of carbobenzyloxy-L-glutamic acid (a-benzyl) -γ-p-nitrophenyl ester (Chem. Ber. 96, 204, 1963) are dissolved in 50 ml of pyridine. The solution is cooled to 0 ° C and within half an hour a solution of 1.25 g (100 mmol) of taurine in 20 ml of water is added dropwise. Then, 3.08 ml (22 mmol) of tri-8 π are added to * 'Ί Γ Γ / 7 Ο U ί \! ·. ι ϋ 'τ j C Ο ethylamine until the mixture, cooling and stirring are interrupted. The mixture is left at room temperature for 72 hours and then evaporated under vacuum. The residue is dissolved in 50 ml of water and IN hydrochloric acid is added to the yellow solution until it is decolorized. To remove the p-nitrophenol, shake the solution ten times with 50 ml of ether each time. This aqueous phase is evaporated under vacuum. 6.9 g of carbobenzyloxy-γ- (α-benzyl) -L-glutamyltaurine-triethylammonium salt are obtained.

The substance is catalytically hydrogenated in the manner described in Example 1b and the solvent is removed by evaporation under vacuum. To remove the triethylamine, the substance is dissolved in a small amount of water and transferred to a "Dowex" 50 x 2 column of 2 x 40 cm.

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Elute with water. "Approximately 120 ml of eluate are collected and then evaporated under vacuum at 35 ° C. Crystallization and isolation of the product are carried out in the manner described in Example 1b. 1.72 g of γ-L glutamyltaurine which corresponds to a yield of 68% relative to taurine, mp 219-220 ° C, = + 14 ° (c = 1, in water).

Example 4 γ-D-Glutamyltaurine

Carbobenzyloxy-γ- (α-benzyl) -Dg-lutamyltaurine-triethyl-ammonium salt prepared in the manner described in Example 3 is catalytically hydrogenated in the same manner as Example 3. The obtained γ-D-glutamyl-taurine melts at 219-220 = -13.9 ° (c = 1, in water).

Example 5 ---------- a) Carbobenzyloxy-YL-glutamyltaurine 529 mg (1.1 mmol) of the carbobenzyloxy-γ- (α-benzyl) -L-glutamyltaurine dissolved in Example 1a in 5 ml of 1N potassium hydroxide solution and leave for four hours at room temperature. Then the solution is shaken three times with 3 ml of ether each time. The aqueous phase is transferred to a 1 x 20 cm 1 x 20 cm column filled with "Dowex" and eluted with water. ("Dowex" is a trademark registered in Denmark). Collect 50 ml of solution which is evaporated to dryness under vacuum at 35 ° C. Crude carbobenzyloxy-35 γ-L-glutamyltaurine is obtained which is purified by paper electrophoresis carried out at pH 6.5. The relative motility to cysteic acid is 1.05%.

Rp (n-Butanol / pyridine / glacial acetic acid / water 15: 10: 3: 12) = 0.57.

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B) γ-L-Glutamyltaurine

The carbobenzyloxy-γ-L-gluta-myltaurine obtained according to Example 5a) is dissolved in 2 ml glacial acetic acid containing 4 moles of hydrocarbon. The mixture is left at room temperature for half an hour and then evaporated at 35 ° C under vacuum. The residue is ripped four times with ether 5 and then separated from the ether by decanting. The obtained γ-L-glu-tamyltaurine is recrystallized in the manner described in Example 3.

Example 6 γ-L-Glutamyltaurine, monosodium salt 10 -----.

Dissolve 25.4 mg (0.1 mmol) of γ-L-glutamyl taurine in 2 ml of water and add 10 ml of 0.01 N sodium hydroxide solution to the solution. The mixture is evaporated at 30 ° C under vacuum. The white crystalline residue is dried in elecsicator over phosphorous toxide.

A monosodium salt of γ-L-glutamyltaurine is obtained. The product has no sharp melting point, begins to shrink at 200 ° C, its color becomes darker, and at approx. 250 ° C char it. The product is equally poorly soluble in methanol and ethanol.

Example 7 ---------- a) Carbobenzyloxy-αL-glutamyl (γ-taurine) glycine ethyl ester 0.48 g (1 mmol) carbobenzyloxy-αL-glutamyl (γ-nitrophenyl ester) ) -glycine ethyl ester (Acta Chim. Acad. Sci. Hung. 65, 375, 2.5 1970) is dissolved in 6 ml of ethyl acetate. The solution is cooled with ice water. At 0 ° C, 0.08 g (1 mmol) of cysteamine in 1 ml of dimethylformamide is first added to the solution, whereupon 0.14 ml (1 mmol) of triethylamine is added dropwise. A precipitate slowly begins to precipitate. The reaction mixture is left for some time in ice water, where it is left at room temperature for one day and finally diluted with a 1: 1 ratio of ether and ethyl acetate. The precipitate is centrifuged and washed first with a mixture of ether and ethyl acetate in a 4: 1 ratio and then several times with ether. After drying over sulfuric acid, the precipitate is washed three times with 1N hydrochloric acid, twice with water, twice with saturated sodium hydrogen carbonate solution.

DK 155433B

10 and finally again twice with water and then dried under vacuum over sulfuric acid. 0.35 g of carbobenzyloxy-cc-L-glutamyl (γ-cysteamine) glycine ethyl ester is obtained, corresponding to a yield of 85%.

Calc'd for C 18 H 25 ° 6 N 3 S = 411.4): C 52.6 H 6.1 S 7.8 Found: C 53.4 H 6.5 S 7.7%

The infrared spectrum exhibited the following maxima for the characteristic groups: NH 3310 cm ”1, C = 0 (C00C₂H ^) 1748 cm” 1, 0 = 0 (Z) 1690 cm ”1, 0 = 0 (amide) 1655 cm” 1 .

Dissolve 100 mg of carbobenzyloxy-α-L-glutamyl (γ-cysteamine) glycine-10 ethyl ester in 2 ml of glacial acetic acid and add 0.5 ml of 30% hydrogen peroxide to the solution. The reaction mixture is left for four hours under ice-cooling. The progress of the reaction is followed by paper electrophoresis. After dilution with water, the reaction mixture is lyophilized. The final product is wound in the form of a foam. 0.11 g (95%) of carbobenzyloxy-α-L-glutamyl (γ-taurine) glycine ethyl ester is obtained.

b) aL-Glutamyl (γ-taurine) glycine 2Q 100 mg of the carbobenzyloxy-αL-glutamyl (γ-taurine) glycine ethyl ester prepared in Example 1a is dissolved in a mixture of 1 ml of trifluoroacetic acid and 1 ml of concentrated hydrochloric acid. The solution is kept for three hours at 35 ° C in a closed bomb tube. The solution is then evaporated in vacuo, the residue is evaporated several times with ether and n-hexane and evaporated again. A white amorphous white substance is obtained which, by electrophoretic examination, appears as a unit and gives a positive ninhydrin spot. The product is α-L-glutamyl (γ-taurine) glycine. Yield 0.06 g (88%).

Calculated for C CH ^NgO₂S (m = 311.3): δ 8 10 · 3 found: S 10.0%.

The infrared spectrum showed the following maxima for the characteristic groups: NH * 3100 cm -1 (wide); OH (C00H) 3200 cm 1 (wide); 0 = 0 (C00H) 1730 cm -1, 0 = 0 (amide-1 1680 cm -1, 0 = 0 (amide-11) 1560; S = 0 (SOpOH) 1220 cm -1 (intensive); S = 0 (SOpO ") 1045 cm" 1

J J

(intensive).

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Hydrolysis: 20 mg of the substance is kept in 1 ml of 6N hydrochloric acid in a molten glass tube for 24 hours at 105 ° C. After cooling, the sample is extracted by electrophoresis at pH 1.8. The solution contains glutamic acid, glycine and taurine.

C) Carbobenzyloxy-α-L-glutamyl (γ-taurine) glycine methyl ester (0.47 g (1 mmol) carbobenzyloxy-α-L-glutamyl (β-nitro-phenyl ester) -glycine methyl ester is dissolved in 6 ml of pyridine. During ice-cooling, 0.125 g (1 mmol) of taurine is first added to 2 ml of water and then 10 o, 28 ml (2 mmol) of triethylamine to the solution in such small portions that the solution remains clear at all times. The reaction mixture is left at room temperature for three days. After evaporation under vacuum, an oil is obtained which, after thorough leaching in ether and then in petroleum ether, is dried under vacuum over sulfuric acid. Carbobenzyloxy-α-L-glutamyl (γ-taurine) glycine methyl ester is obtained.

d) αL-Glutamyl (γ-taurine) glycine 100 mg of the carbobenzylyloxy-αL-glutamyl (γ-taurine) glycine methyl ester prepared in Example 7c are reacted with 4 ml of 2N acetic acid hydrochloric acid at room temperature until combined amount of substance dissolved (about 30 minutes). The obtained clear solution is precipitated in 30 ml ether and then left in a cold place for a day. The resulting precipitate is centrifuged, washed several times with ether and then dried under vacuum over potassium hydroxide, sulfuric acid and phosphorus pentoxide. The electrophoretic study shows that the hydrogen bromide of the α-L-glutamyl- (γ-taurine) glycine methyl ester is recovered in practically pure form.

The obtained salt is reacted under cooling with ice water for three hours with 2 ml of N sodium hydroxide solution. The progress of hydrolysis is followed electrophoretically. The reaction mixture is subjected to ion exchange with 10 ml of "Dowex" 50 in H + form and then lyophilized. Since the electrophoresis still shows contaminants, the product is recrystallized several times from aqueous ethanol until a corresponding purity is obtained. 40 mg (59%) of α-L-glutamyl (γ-taurine) glycine is obtained.

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DK 155433 B

The infrared spectrum exhibits the following maxima for the characteristic groups: NH 3310 cm "" 1; NH * 3100 cm 1 (wide; 0 = 0 (COOH) 1730 cm -1; C = 0 (amide i) 1650 cm -1) C = 0 (amide II) 1570 cm -1; S = 0 1220 (intensive) , 1045 cm -1 (intensive).

E) Y-L-Glutamyltaurine 100 mg of it under cl. α + b or c + d in this example, α-L-glutamyl (γ-taurine) glycine is dissolved in 25 ml of a 0.2 molar ammonium hydrogen carbonate buffer having a pH of 8.5. To the solution is added 1 mg of carboxypeptidase A dissolved in 0.5 ml of water. J Manufacturer of the enzyme: Serva,

Heidelberg). The mixture is kept for 24 hours at 37 ° C in thermostat and lyophilized. In the dried lyophilisate, Ύ-L-glutamyltaurine and glycine can be detected. The pure γ-L-gluta myltaurine can be obtained by electrophoresis or chromatography on a Dowex 50 column.

Example 8 a) Carbobenzyloxy-γ- (α-benzyl) -L-glutamylhomotaurine 1.082 g (2.2 mmol) of carbobenzyloxy-L-glutamic acid (α-benzyl) -yp-nitrophenyl ester is dissolved in a mixture of 6 ml. pyridine and water in a 2: 1 ratio. To the solution is first added 278 mg (2 mmol) of homotaurine and then 0.59 ml (4.2 mmol) of triethylamine. The yellow solution is left at room temperature for 72 hours and then evaporated under vacuum. The oily residue is dissolved in water, neutralized with hydrochloric acid and then extracted to remove p-nitrophenol in a continuous ether extractor for eight hours. The aqueous phase is evaporated under vacuum. 1.68 g of carbobenzyloxy-γ- (α-benzyl) -L-glutamyl homotaurine are obtained.

B) γ-L-Glutamylhomotaurine

The total amount of the substance prepared according to Example 8a) (1.68 g) is dissolved in 10 ml of 50% aqueous ethanol, to which 0.3 g of 10 µl palladium activated carbon is added and hydrogen is passed through the suspension for four minutes. hours. The solution is then filtered and evaporated under vacuum. The residue is dissolved in 1-2 ml of water and to remove

DK 155433 B

13 ethylamine is added to a 1 x 35 cm column with "Dowex" 50 x 2 in H + form. Elute with water. 50 ml of eluate are collected and evaporated under vacuum. As the residue, 4Ί ml of γ-L-glutamyl homotaurine is obtained, which corresponds to a yield of 82%. The electrophoresis carried out at pH 6.5 exhibits a small amount of contamination 5 of partly neutral and partly acidic compounds (homotaurin or glutamic acid). For example, the product may be purified by preparative electrophoresis.

Both by electrophoresis carried out at pli 6.5 and by electrophoresis carried out by arrow 1.8, the compound migrates in the direction of the cathode. The mobility with respect to cysteic acid is at pH 6, 1q 0.68, at pH 1.8 0.50.

Rp (n-Butanol / pyridine / glacial acetic acid / water 15: 10: 3: 12) = 0.19 Example 9 a) Carbobenzyloxy-Y- (α-benzyl) - L-glutamyl-N-methyltaurine 1.083 g (2 (2 mmol) carbobenyzloxy-L-glutamic acid (α-benzyl) -yp-nitrophenyl ester is reacted in the manner described in Example 8a) with 278 mg (2 mmol) of N-methyltaurine. 1.59 g of carbo-benzyloxy-γ- (α-benzyl) -L-glutamyl-N-methyltaurine are obtained.

B) Y-L-Glutamyl-N-methyltaurine

The total amount of the substance (1.59 g) prepared according to Example 9 (a) is catalytically hydrogenated in the manner described in Example 8 (b). 423 mg of γ-L-glutamyl-N-methyltaurine is obtained, which corresponds to a yield of 79%. Both at pH 6.5 and at pH 1.8, the compound migrates by paper electrophoresis in the direction of the cathode. Mobility to cysteic acid: pH 6.5: 0.68; pH 1.8: 0.49.

• (n-Butanol / pyridine / glacial acetic acid / water 15: 10: 3: 12) = 0.16.

Example 10 ----------- a) Carbobenzyloxy-γ- (α-benzyl) -L-glutamyl-L-cysteic acid 2.87 g (6.6 mmol) of carbobenzyloxy-L-glutamic acid ( The α-benzyl) -yp-nitrophenyl ester is dissolved in 20 ml of pyridine and a solution of 1.25 g (6 mmol) of L-cysteic acid monohydrate is added in a mixture of 17 ml of water and 17 ml of pyridine. After adding 2.6 ml - -

DK155433B

14 (18.6 mmol) of triethylamine leaves the reaction mixture at room temperature for 72 hours. The solution is then evaporated under vacuum at 30 ° C. The residue is dissolved in 20 ml of water, acidified with concentrated hydrochloric acid and then shaken 15 times with 10 ml of ether each time. The aqueous phase is evaporated under vacuum at 35 ° C. Carbobenzyloxy-γ- (α-benzyl) -L-glutamyl-L-cysteic acid is obtained.

b) T-L-Glutarnyl-L-cysteic acid

The product prepared according to Example 10a) is dissolved in 20 ml of water, 0.3 g of 10% palladium-activated charcoal is added and hydrogen is passed through the suspension for three hours. The reaction mixture is worked up in the manner described in Example 8 (b). Γ-L-glutamyl-L-cysteic acid is obtained which melts at 187 ° C. Relative to cysteic acid, the mobility of paper chromatography is: at pH 6.5: 1.21; at pH 1.8; 0.54.

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Example 11 a) Carbobenzyloxy-γ- (ct-benzyl) -L-glutamylcolamine phosphate is dissolved in 1.083 g (2.2 mmol) of carbobenzyloxy-L-glutamic acid (a-benzyl) -yp-nitrophenyl ester in 6 ml of a prepared mixture of pyridine and water in a 2: 1 ratio. To the solution is first added 282 mg (2 mmol) of colamine phosphate (U.S. Patent No. 2,730,542) and then 0.87 ml (6.2 mmol) of triethylamine. The reaction mixture is left at room temperature for 72 hours and then evaporated under vacuum. The further work-up is carried out in the manner described in connection with carbobenzyloxy-γ- (α-benzyl) -L-glutamylhomotaurine (Example 8a). 1.25 g of carbobenzyloxy-γ- (α-benzyl) -L-glutamyl-cholamine phosphate are obtained.

b) T-L-Glutamylcolamine phosphate

The total amount of the product obtained in Example 11a (1.25 g) is catalytically hydrogenated to remove the protecting groups. The hydrogenation as well as the purification by ion exchange is carried out in the manner described in Example 12b in connection with the preparation of γ-L-glutamyl homotaurine. 470 mg of γ-L-glutamylcolamine phosphate is obtained, which corresponds to a yield of 91%. However, it is evident from paper electrophoresis that the product contains a contaminant.

DK 155433 B

15 purification of approx. 15-20% of colamine phosphate. As a cleaning method comes, among other things. electrophoresis on speech.

In paper electrophoresis, the compound migrates to the cathode at both pH 6.5 and pH 1.8. The motility relative to cysteic acid is: pH 6.5: 0.75; pH 1.8: 0.36.

Rp (n-Butanol / pyridine / glacial acetic acid / water 15: 10: 3: 12) = 0.18.

Example 12 a) Carbobenzyloxy-β- (a-benzyl) -L-aspartyl taurine 526 mg (1.1 mmol) of carbobenzyloxy-L-aspartic acid (a-benzyl) -p-nitrophenyl ester (Chem. Ber. 97, 1789 , 1964) is dissolved in 5 ml of pyridine. The solution is cooled to 0 ° C, and in small portions a solution of 125 mg (1 mmol) of taurine is added in 2 ml of water followed by 0.28 ml (2 mmol) of triethylamine. The reaction mixture is left at room temperature for 48 hours and then evaporated under vacuum. The residue is dissolved in 5 ml of water and to the initially yellow solution an N hydrochloric acid is added until the solution is decolorized. To remove ρ-nitrophenol, the solution is shaken ten times with 5 ml of ether each time. The aqueous phase is evaporated under vacuum. 20478 g of carbobenzyloxy-β- (a-benzyl) -L-aspartyl taurine are obtained.

b) β-L-Aspartyl taurus

The total amount of the product obtained in Example 12a) is dissolved in 6 ml of 50% aqueous ethanol. To the solution is added 100 mg of 10% s of palladium-activated charcoal, which is hydrogenated during the introduction of hydrogen gas for four hours. After filtration and evaporation in vacuo, the triethylamine is removed from the product by ion exchange in the manner already described in connection with the preparation of γ-L-glutamylhomotaurine in Example 8 b). 172 mg of β-L-30 aspartyl taurine is obtained, which corresponds to a yield of 91%. The product is contaminated with a small amount of taurine. can be removed by electrophoresis.

In paper electrophoresis, the compound migrates toward the cathode at both pH 6.5 and pH 1.8. Movement relative to cysteine stearic acid: pH 6.5: 0.77; pH 1.8: 0.58.

Rp (n-Butanol / pyridine / glacial acetic acid / water 15: 10: 3: 12) = 0.16.

Example 13

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16 a) Carbobenzyloxy-β- (α-benzyl) -L-aspartyl homotaurine 526 mg (1.1 mmol) of carbobenzyloxy-L-aspartic acid (α-benzyl) -p-nitrophenyl ester and 139 g (1 mmol) of homotaurine are reacted in the manner described in Example 12 (a). Carbobenzyloxy-β- (α-benzyl) -L-aspartylhomotaurine is obtained.

b) β-L-Aspartyl homotaurin

The product obtained according to Example 13a) is catalytically hydrogenated in the manner described in Example 8. b). 203 mg (84/0 β-L-aspartylhomotaurine) is obtained.

In paper electrophoresis, the compound migrates at both pH

6.5 and at pH 1.8 in the direction of the cathode. The motility relative to cysteic acid is: pH 6.5: 0.72; pH 1.8: 0.53.

R R (n-Butanol / pyridine / glacial acetic acid / water 15: 10: 3: 12) = 0.17.

Example 14 a) Carbobenzyloxy-β- (o-benzyl) -L-aspartylcolamine phosphate 2 Carbobenzyloxy-L-aspartic acid (α-benzyl) -β-ρ-ηΐtrophenyl ester and colamine phosphate are reacted with each other in the manner described in Example Ha) . Carbobenzyloxy-β- (α-benzyl) -L-aspartyl-colamine phosphate is obtained.

b) β -L-Asparty cholesterol phosphate 25 -;

The substance obtained in Example 14 a) is catalytically hydrogenated in the manner described in Example 8b). Β-L-aspartyl-colamine phosphate is obtained.

In paper electrophoresis, the compound migrates at both pH 3Q 6.5 and pH 1.8 toward the cathode. Mobility relative to cysteic acid: pH 6.5: 0.81; pH 1.8: 0.40.

Rp (n-Butanol / pyridine / glacial acetic acid / water 15: 10: 3: 12) = 0.14.

35

Example 15

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17 T-L-Glutamylcolamine Phosphate In the manner suitable for the preparation of glutamic acid γ-amides (Acta Chim. Acad. Sci. Hung. 64, 285, 1970), lcarbc 5 benzyloxy-γ- (α: -benzyl) -L-glutamylcolamine is prepared. Dissolve 4.14 g of this product (10 mmol) in 50 ml of absolute pyridine and add 9 g (33 mmol) of diphenylphosphoric acid chloride. The reaction mixture is kept at 0 ° C for twelve hours and then diluted with 80 ml of chloroform. The separated product is filtered off, washed with hydrochloric acid and then with 10 water and finally dried in desiccator over potassium hydroxide. Dissolve the obtained carbobenzyloxy-γ- (α-benzyl) -L-glutamylcolamine phosphate: in 15 ml glacial acetic acid containing 3.3 mol / l hydrochloric acid and leave the solution for 15 minutes, then evaporate in vacuo at 25 ° C. The residue is dissolved in 30 ml of 1N sodium hydroxide and the solution is left at room temperature for one hour. Then the pH = 4 is adjusted with acetic acid and to remove phenol and benzyl alcohol, the solution is extracted three times with 30 ml of ether each time. The aqueous phase is transferred to a column of "Dowex" 50 in H + form and eluted with water. The eluate is evaporated under vacuum 20 and the residue is recrystallized from a mixture of acetone and water in a 2: 1 ratio. 0.8 g of γ-L-glutamylcolamine phosphate is obtained

DK 155433 B

18

Biological Test Report A.

Study of γ-L-glutamyltaurine as in this report is called litoralon.

5 In the following Tables 1-6, the test results are shown as mean values + standard deviation with the number of brackets. Determination of whether there is a significant difference between control trials and trials in treated animals was done with Student's t-test.

10

Table 1

Effect of Litoralon on vitamin A concentration in serum.

Group of Litoralone Vitamin A concentration in serum 15 µg / day_ I. Control - 28.3 + 0.7 (20) II. 0.1 41.9 + 1.0X (20) III. 0.3 32.9 + 1.1XX (20) IV. 1.0 27.4 + 0.6 (20) 20 - x PK 0.001 xx P <0.01 20 Sprague Dawley rats (10 males and 10 females) weighing 25 180-200 g were treated orally with the daily indicated in Table 1 do see litoralon in the form of an aqueous solution for a period of 8 days. On the 9th test day, the animals were sacrificed by decapitation and the blood was collected. The vitamin A concentration in the serum was determined by Neeld and Pearson's method.

30 35 19

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Table 2

Effect of Litoralone and Vitamin A on Granule Conversion Caused by Implanted Cotton Balls _; _; __ 5 Group Dose Dry Weight of

Vitamin Ax litoralon granuloma local local oral g mg µg yg / day I. Control - - - 52 + 1.0 (24} 1q II.Control +

Solvent - - - 54 + 3.1 (8) III. 2 - 64 + 2.5 (8) IV. 2 0.1 - 65 + 2.7 (8) V. - 0.1 73 + 2.9 (8) 15 vi. 2 - 0.1 90 + 4.1 (8) x Hoffmann La Roche

The difference is significant: between groups II and III at 20 P <0.05, between II and V at P <0.001, and between V and VI at P <0.01. Granule formation was determined according to Lee et al with male Sprague-Dawley rats weighing 110-120 g. The tampons were removed from the dorsolateral subcutaneous implants after 10 days and weighed after drying to constant weight at 65 ° C.

25 30 35

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DK 155433 B

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22

The effect of Litoralon on the metamorphosis of Rana dalmatina

Group Body length Tail length _ mm_mm_ 5 Control 45.9 + 0.2 31.4 + 0.2 (60)

Treated animals 40.2 + 0.2X 26.7 + 0.2X (60) x Significance: P <0.001 1 q The experiment was conducted with larvae of Rana dalmatia with a age of 30 days, a body length of 20-25 mm, and with already visible projecting hind legs. Over a period of 30 days, the test animals were placed in tap water containing 0.5 µg / ml litoraion for two hours each day. The control group was placed in 15 tap water without the addition of litoraion. The tails were measured on the 30th day.

Table 6

Litoralone's effect on blood sugar levels 20

Group I. Study II. Survey _mg% _mg% _

Control 94 + 3.6 (10) 94 + 4.09 (10)

Treated animals 82.4 + 3.8 (10) 81 + 1.32 (10) 25 Significance at I. study: P <0.05 Significance at II. study: P <0.01

Male CGY white body rats 160-180 g were used for the experiment. The animals were kept on a standard diet. Blood samples were taken on the 5th day of testing after 18 hours of fasting. The blood sugar cane meal was carried out according to the method of E. Hulman. Litoralon was given orally at daily doses of 1 µg / kg.

35

DK 155433 B

23

Biological Test Report B.

Study of synthetic γ-aspartyl taurine and its derivatives.

5 1. β-Aspartyl-N-methyltaurine a) Effect on blood sugar level

Control 107 mg%

Treated animals 93 mg%

Significance: P <0f05

20-20 rats were used for the samples. The measurements were carried out after 18 hours of fasting. The dose used was 1 µg / kg body weight for 4 days in the form of a solution by oral administration.

b) Effect of increasing serum vitamin A level 15 Oral dose Induced vitamin A yg% yg / 200 g body weight 0 (control) 8.5 5 11.0 1 11.5 20 0 ^ 3 12.5 0.1 16.1 0.05 14.8 0.01 12.5 0.005 10.5 Significance: P <0.01

For the experiments, 20-20 Wistar male rats weighing 200-220 g were used.

Trial period: 6 days.

C) Effect on blood silicon level:

Silicon (mg / g blood) _ 0 hours_2® days_40 days

Control group H0.0 © 4 0.120 + 0, © Sat 0.154 + 0.015

Treatment group 35 µg / day 0.100 + 0.005 0.315 + 0.014 ** 0.345 + 0.015

Treatment Group View II 10 µg / day 0.107 + 0.009 0.370 + 0.119 ** 0.360 + 0.017 **

DK 155433 B

24

Results are significant from the 13th day at the significance level P <0.001 and from the 20th day at the level P <0.001. The experiments were performed on inbred male rabbits of body weight 2.5-3 kg. The active ingredient was administered orally in the 5 daily doses shown in the table. The determination of silicon was performed according to Gaubatz's method (Gaubatz E.f. Clin. Wschrft. 14, 1753, 1935) in 5 ml of blood samples taken from the animals' ears.

d) The effect of β-aspartyl-N-methyl-taurine and vitamin A on the .jq granulogenic effect caused by implantation of cotton wool;

Group Dose Weight of dry

Vitamin Ax (3-aspartyl-N- rf 9ranuloma methyltaurine local local oral mg gig / day ^ Control I. - - - 54 + 1.7

Control II. + solvent - - - 55 + 3.4

Treatment Group III. 2 - - 66 + 12.5

Treatment Group IV. 2 0.1 - 67 + 2.6 Treatment Group V. - - 0.1 79 + 2.8

Treatment Group VI. 2 - 0.1 96 + 4.4 x Hoffmann la Roche

The differences are significant as follows: 25 Between groups II and III P <0.05, between groups II and V P <0.001 and between groups V and VI P <0.01.

The determination of granuloma was performed on male Sprague-Dawley body weights 110-120 g by the method of Lee et al (Lee K.H., Fu Ch.Ch., Spencer M.R. Tong T.G. and Poon R.J., Pharm.

3Q Sci., 62, 895, 1973). The dorsolaterally subcutaneously implanted tampons were removed after 10 days and measured after drying at 65 ° C to constant weight.

2. β-Asparfeyl homotaurin.

25 a) Effect on blood sugar level

Control group 105 mg%

Treated group 94 mg%

Significance, number of test animals and test method were as indicated

DK 155433 B

25 b) Increasing effect on serum vitamin A level

Oral dose Induced vitamin A

yg / 200 g body weight yg% 0 (control) 8.6 5 5 12.0 1 13.5 0.3 14.0 0.1 15.8 0.05 15.0 10 0.01 12.0 _0, 005_10,0_

Significance, number of test animals and test method were as indicated above under section. Ib).

C) Effect on blood silicon level;

Silicon (mg / g blood) _0 hours_20 days_40 days

Control group 0.104 + 0.009 0.134 + 0.015 0.157 + 0.020 on Treatment group I. 5yg / day 0.094 + 0.007 0.309 + 0.014 0.340 + 0.014 '

Treatment Group II. 10 µg / day 0.109 + 0.010 0.372 + 0.120 0.363 + 0.0185 ^

Significance, number of test animals, arrangement and method of determination as indicated above under cl. 1 c).

25 d) The effect of β-aspartyl homotaurin and vitamin A on the granulomatous effect of cotton implantation: ____

Dose Weight of dried 3Q Vitamin Ax β-aspartyl granuloma local homotaurin g mg local oral ______g / day_

Control group I - ~ - 52 + 1.5

Control group II - - - 53 + 3.2 + 35 + solvent

Treatment Group III. 2 - - 64 + 12.3

Treatment Group IV. 2 0.1 - 65 + 2.4

Treatment group V. - - 0.1 77 + 2.6

DK 155433B

26 x Hoffmann la Roche

Significance, number of test animals, arrangement and method of determination were as indicated above under point (ld).

3. γ-Glutamyl-Colamine Phosphate.

a) Effect on blood sugar level

Control group 104 mg%

Treatment group 93 mg%

Significance, number of test animals, arrangement and dosage were as indicated above under point 1a).

b) Increasing effect on serum vitamin A level

Oral dose of Vitamin A action ^ yg / 200 g body weight yg% 0 (control) 8.8 5 12.3 1 13.4 0.3 14.3 20 0/1 16.0 0.05 15.5 0, 01 11.2 0.005_9j_2_

Significance, number of test animals, arrangement and method of determination were as indicated above under point 1 (b).

c) Effect on blood silicon level;

Silicon (mg / g blood) 0 hours 20 days 40 days 30-2-2-

Control group 0.106 + 0.011 0.136 + 0.017 0.159 + 0.022

Treatment group I 5 µg / day 0.096 + 0.009 0.311 + 0.016 0.340 + 0.017

Treatment group II 10 µg / day 0.111 + 0.011 0.374 + 0.12lxx 0.365 + 0.019 55 Significance, number of test animals, arrangement and method of determination were as given above under item 1 (c).

DK 155433 B

(D) the effect of γ-glutamyl-colamine phosphate and vitamin A on the granuloma-inducing effect of water implantation;

Dose Weight of dried granuloma

Vitamin Ax γ-glutamyl mg 5 Local Colamine Phosphate mg Local Oral _µg µg / day_

Control group I. - - - 49 + 1.2

Control group II. + solvent - - - 50 + 2.9 10 Treatment group III. 2 - - 61 + 12.0

Treatment Group IV. 2 0.1 - 62 + 2.1

Treatment group V. - - 0.1 74 + 2.3

Treatment Group VI. 2 - 0.1 91 + 3.9 x Hoffmann la Roche

Significance, number of test animals, arrangement and method of determination were as indicated under 1 d) above.

20 25 30 35

Claims (4)

5. I <| H2> n 1 CO-N-CH- (CH,) .- B1 I12 2 * RR 1. where is a group of the formula -SO2OH or -OPOfOH ^ / R is a hydrogen atom or an alkyl group having 1-4 carbon atoms, 2R is a hydrogen atom or a carboxyl group, n is 1 or 2 and 15h is 1 or 2, or physiologically acceptable salts or optically active antipodes thereof, characterized in that 4 a) the protecting group R in a racemic or optical active compound of the general formula 4 R-NH-CH-COOH (f2> n HA CO-N-CH- (CHO).-B1 II 2 t 25. R1 wherein 4 R is an alkoxycarbonyl group having 1-4 carbon atoms in the alkoxy moiety, an aralkoxycarbonyl group having 7-9 carbon atoms in the aralkoxy moiety, or a phenoxycarbonyl group optionally having a halogen, alkoxy or nitro substituent in the phenyl ring, and Rr R, B, n and t have the above meanings, are decomposed by acidolysis, hydrogenolysis, sodium treatment or diluted ammonium hydroxide treatment, or b) a racemic or optically active compound of the general formula HoN-CH-COA3 5 I 5 (CHp). | 2 n IIB CO-N-CH - (CHO) -B1 M2 2 * RR 10 wherein 3A is an alkoxy group of 1-4 carbon atoms, an aralkoxy group of 7-9 carbon atoms, or a group of the general formula - (NH-CH-CO) -Y 1. In 5 Γ 15 rd 5 where R is a hydrogen atom, an alkyl group, an aralkyl group, a hydroxy-substituted aralkyl group or a heteroaryl alkyl group, Y is a hydroxy group, an alkoxy group having 1-4 carbon atoms 20 or an aralkoxy group having 7- 9 carbon atoms, r is an integer from 1-10, and 2 1 R, R, B, n and t have the meanings given above, are subjected to acidolysis, hydrogenolysis or enzymatic hydrolysis, or c) the protecting groups attached to the α-amino group and the α-carb oxyl group in a racemic or optically active compound of the general formula R 4 -NH-CH-CO-A3 I (<j: H2) n IIC CO-N-CH- (CHJ. -B1 I \ 2 2t RR n j- 2 4 3 1 ό ~> wherein R, R, R, A, B, n and t have the above meanings, or in a salt thereof, are simultaneously decomposed by acidolysis, alkaline hydrolysis, hydrogenolysis or treatment with I Sodium and, if desired, converting a compound obtained by any of the methods a) -c) into a physiologically acceptable salt which of or releasing the compound from a salt and / or preparing a won compound in an optically active form by cleavage of a won racemic product. 10 15 20 25 35