NO146430B - ANALOGY PROCEDURE FOR THE PREPARATION OF NEW THERAPEUTICALLY ACTIVE AMINO ACID DERIVATIVES - Google Patents

Description

The present invention relates to an analogue method for the production of new, therapeutically active amino acid derivatives. The process compounds have valuable biological and pharmaceutical effects.

The amino acid derivatives produced according to the invention appear in the introduction to the claim, and the methods used are specified in the characteristic of the claim.

Among the process compounds, due to their biological activities, Y-L-glutamyl-taurine in particular must be highlighted, which compound has the formula:

and which has a broad therapeutic and preventive effect spectrum for morbid changes that are indirectly or immediately traceable to damage by "AGAS" (aerobiospheric genetic adaptation system).

To explain the term "AGAS", the most important tissues and organs that form this system are listed below.

a) All biological interfaces that are in contact with the external air such as the biosphere (skin and skin tissue, Cornea and

Conjunctiva, mouth and throat cavity, respiratory tract and lungs);

b) Skeleton and limbs (tube bones and spongy joints, ball joints, synovial membrane, skeletal muscles); c) The organs that take part in the regulation of ion skin posture (transepithelial transport system: intestinal tufts and renal channels); d) The thecodont (in the dental alveoli with roots attached) set of teeth necessary for the comminution of food; e) Hearing, smelling and vocal organs.

The process compounds thus exert a beneficial biological or therapeutic effect on the organs or tissues of the AGAS system listed here.

The process compounds further act on the following functions that are related to the AGAS system: Radiation protection effect, favors wound healing, general activating effect on the Mesenchyma, protection against the ever-increasing risk of infection and soiling of the skin and mucous membranes (Lysozyme formation on the moist mucous membrane , activation of ciliated epithelia in the respiratory tract, etc.), increased protection against infections caused by viruses and fungi.

Against the constantly and highly increasing effects of stress (e.g. metrological influences, strong differences between day and night temperatures, increased risk of injury) in life on the mainland, the procedure compounds are effective as they stabilize the adaptation syndrome and at the same time prevent peripheral tissue damage from glucocorticoids (such as damage to the connective tissue, damage to the bone matrix, etc.). Development of immune homeostasis (increased recognition ability of the body, for which cells are body-specific and

which ones aren't).

The process compounds exert their effect partly immediately, partly over the control of vitamin A metabolism, by formation of vitamin A metabolites giving a stronger polar character. This effect is comparable to that of parathyroid hormone on the 25-hydroxy-cholecalciferol-1α-hydroxylase enzyme in the kidney channels. With this explanation, the broad pharmacological, biochemical and therapeutic action of the method compounds becomes understandable. The directions of action of the compounds are as follows:

A) Effects with vitamin A grade:

a) Pharmacological and biochemical effects: Labeled sulphate is incorporated to a high degree in the sternal cartilage of rats, respectively the eye lens, liver and lung tissue of chicken foetuses; radio-actively labeled phosphorus is incorporated to an increased extent in the sternal cartilage of rats; chondroitin sulfate synthesis-increasing effect; beneficial effect on wounding, respectively the experimentally reduced wounding in rats and dogs by the administration of cortisone; potential effect on the action of vitamin A on experimentally induced hypo- and hypervitaminoses in rats and dogs; attenuating effects on ulcer-induced stress effects in rats; beneficial effect on degranulation of mast cells; increasing effect of the production of Lysozyme; effect on the household of the trace elements (silicon, zinc, copper, manganese, fluorine); promoting effect on epithelial formation; increasing effect of the alkaline phosphatase activity; it exerted an effect on the granuloma sac formation induced by local action of vitamin A; the very flat course of the dose-effect curve or the change in sign of the effect at large doses; activating effect on the golgi-1 apparatus; beneficial effect on the formation of goblet cells; increasing effect on the concentration of vitamin

A.

b) clinical-therapeutic effects: keratocunjunctivis sicca; Sjogren's syndrome; rhino-laryngo-pharyngitis sicca;

ozaena; chronic bronchitis, sinobronchitis; Mucoviscidosis; constitutional lung diseases in young children; periodontal disease; susceptibility to infection of the skin and mucous membranes against viruses and fungi; cortisone-antagonistic action; beneficial effect on dressings for surgical wounds and mucosal wounds; erosion colli; pruritus-like; impairment of the sense of smell and taste.

Effect without vitamin A grade:

a) Pharmacological and biochemical effects: Effect on the blood sugar level in the form of the temporary lowering;

increasing effect on phosphaturia, lowering effect on the phos mirror in serum; radiation protective effect; in maze trials with inactive animals reduces the time required to reach the goal; impairing effect on experimentally induced fluoride and cadmium toxicosis; increasing effect on the cyclic adenosine monophosphate depletion of the kidneys; limiting effect on the symptoms of experimental

tent induced Lathyrismus; reduction of histamine sensitivity; increasing effect on the activity of the liver enzyme Tyrosinaminotransferase. b) Therapeutic effects: weak radiation damage; viti ligo; muscle hypotonia; psychoenergetic effect;

beneficial effect on involutional and gerontological conditions as well as on mnestic functions; keloid tendencies; Spondylosis ankylopoetica; on wear-and-tear disorders of the locomotor organs; sclerotic fundus; amyloidosis;' morphea; fibrocystic mastopathy.

When administering the process compounds, the duration of treatment is very different. Many diseases (e.g. Thino-laryngo-pharyngitis sicca) become symptom-free by oral administration of 3 x 5 micrograms daily already after 2 weeks, for symptomatic improvement of other diseases (e.g. periodontitis, Sjogren's syndrome) is 1 to 2 months necessary, in case of enough other diseases (e.g. spondylosis ankylopoetica) and the treatment is continued for 1/4 to 1/2 year.

From the process compounds, pharmaceutical, preventive, cosmetic or veterinary medicinal preparations can be easily prepared as desired. The preparations may contain a single active substance or a combination of active substances.

The dose of pure active ingredient amounts to 50 - 500 ng per day and kg body weight and is administered in three single doses.

A tablet contains 2 - 20 µg, preferably 10 µg of active substance and also biologically inert carriers (e.g. milk sugar, starch) as well as the usual tableting aids (granulating agents and lubricants, such as polyvinylpyrrolidone, gelatin, talc) , magnesium stearate, "Aerosil" etc.).

Because of the extremely small doses, it is appropriate to add the active substance to the tablet mass already before granulation in the form of a solution. In this way, the active substance is evenly distributed. The small active ingredient content also makes it possible, when many millions of tablets are produced annually, to produce these in a large laboratory and then at a price that is acceptable to every patient. The active ingredients are stable, the tablets can therefore be placed on the market without specifying a deadline for use. The active substance content of prolonged-acting tablets or spansular capsules can be 10 - 20 ug. In the case of injectable preparations, the appropriate dose per ampoule 5-10 Ug, and the preparation can optionally be prepared as powder ampoules, in which the active ingredient is mixed with an indifferent, water-soluble diluent. The parenteral administrations can be done intramuscularly, subcutaneously or intravenously. In the specified concentration, the active substances damage neither tissue nor vessel walls. The active substances can also be administered as an infusion. Suppositories contain 2-20, preferably approx. 10 Mg of active substance and is produced from cocoa butter or from synthetic waxes or fats (e.g. the "Imhausen-Masse" produced in the Federal Republic of Germany) which are suitable for this purpose. The active substance content of cosmetic or skin conditioning ointments amounts to 0.1-1 yg/g. Ointment base can be hydrophilic or hydrophobic and contains the usual ingredients: e.g. cholesterin, paraffin, glycerol, lanolin, cocoa butter, linseed oil, etc. The active substances can further be processed as aerosol preparations, as the active substance content is also 0.1 - 1 ug/g - When processed as perlingual tablets, one tablet contains 10 ug of active substance, and the dissolution time of the tablet is at \ - 1 hour. Polymers to retard the release of the active ingredient can, for example, are prepared as suspensions and contain 1-5 Ug' of active substance per g polymer. Injection preparations with prolonged action can either be prepared using high-molecular polymers or from the salts formed with high-molecular organic bases (e.g. Histone, Protamine) of the process compounds, one ampoule containing 10 - 20 Ug of active substance. Powders for cosmetic or skin application purposes are prepared in the usual carriers (e.g. talc) and contain 0.1 - g of active ingredient per powder. For eye treatment, the compounds are prepared as drops or as ointments which are miscible or not with the tear fluid; and the active substance content of these preparations amounts to 0.1-1 Ug/g. Children should be given the process compounds in a dosage of approx. 0.3 yg per kg body weight.

The sterile preparations are suitably prepared by sterile filtration.

The desired preventive pharmacological or cosmetic effect of the preparations listed above can be increased and extended by numerous combinations. As possible biologically active additives, the following explicitly mentioned are primarily used: Vitamins A, C, E and K, trace elements, cortisone and its derivatives, progesterone, thyroid hormones, radio-mimetic and immunosuppressive active substances, psychopharmaca, especially psychosedatives, timoleptica . alone.

The process compounds can also be used as an additive to foodstuff or drug premixes. They partly cause weight gain, partly reduce the need for vitamin A or improve its metabolism. Furthermore, the compounds increase the resorption of the trace elements, their mirror in the blood is raised. When using the compounds as an additive to animal feed, the appropriate oral dosage amounts to 200 ng/kg per day. Mixed in the feed, this roughly corresponds to a concentration of 1 - 2 ug/kg, respectively 1-2 mg/barrel of feed, i.e. a concentration of 0.001 - 0.002 ppm. In light of these very low concentrations, the use as a precursor additive is extremely economical. Preferably, the compounds are mixed into vitamin premixes or used in microcapsules, which, in addition to the process compounds, contain other necessary precursor additives. The compounds can also be used in the drinking water, in the lick salt or possibly also as a spray.

In veterinary medicine, the process compounds have similar areas of application as in human medicine, i.e. e.g. skin damage (scaling), wounding, broken bones, etc.

It is a common structural property of all the compounds with the general formula (I) that they contain a dicarboxylic acid substituted in the α-position or its derivative also further substituted in other places, above whose w-carboxyl group with an acid amide bond is bound a primary or secondary alkylamine, which, in addition to various substituents on its alkyl side chains in the w-position, contains a group of strongly acidic character.

The object of the present invention is analogous methods for the preparation of the compounds of the general formula (I), its salts and optical isomers. The compounds are produced according to the invention as stated in the characteristics of the claim.

In the detailed description of the embodiments of the present method, the method variant i) will first be mentioned, according to which the compounds of the general formula (I) are prepared by forming an acid amide bond. In this case, one on the amino or on the anino and α-carboxyl group with a protecting group, respectively several protecting groups,

or also with other groups substituted derivative of an α-aminodicarboxylic acid over its u>-carboxyl group linked e.g. to 2-aminoethanesulfonic acid, 3-aminopropanesulfonic acid or 2-phosphonoethanolamine. When designing the acid amide bond, different protecting groups can be used. The most suitable coupling method is the active ester method (monograph for the formation and selective removal of protecting groups and coupling methods: E. Schroder, K. Liibke: The Pepl^.des, Vol.1: Methods of peptide synthesis, Academic Press, 1965) .

According to variant h) of the present method, one can also proceed by acylating cystamine or its substituted derivatives on the amino group with α-aminodicarboxylic acid derivatives. The acylation of cystamine can be carried out by different methods (the active ester method, the mixed anhydride method). The compounds thus obtained can according to method variant

f) is reacted with hydrogen peroxide or peracids, whereby the disulphide bond is split oxidatively and a compound is formed with the

general formula (I).

Likewise, process variant d) leads to a compound with the general formula (I) in that the o-amide is first prepared from an o-aminodicarboxylic acid, which in the amine part instead of a strongly acidic functional group contains another functional group, e.g. a sulfhydryl or sulfinic acid function. From these compounds, the desired compounds are produced by oxidation. If the functional group is a halogen atom or a p-toluenesulfonyl group (J. Chem. Soc. 824, 1964), substitution with alkali sulphite or alkali hydrogen sulphite gives compounds of the general formula (I), which contain a sulpho group. Compounds with the general formula (I), which contain a hydrogen sulfate or dihydrogen phosphate group, are obtained by esterification of hydroxyl group-containing compounds. If the starting compound contains a sulphonic acid ester group, the compounds of the general formula (I) are obtained by gentle partial hydrolysis.

Furthermore, one can proceed from glutamine or asparagine or their substituted derivatives (with the exception of the acid amide). In this case, according to process variant e) an acid amide hydrogen atom which has an acidic character is substituted with metallic sodium and the compound thus obtained is converted by reaction with 2-bromoethanesulfonic acid or its salts or alkenesulfonic acid into a compound with the general formula (IN). As starting compound, w-amides of α-aminodicarboxylic acids are used, e.g. u)-vin<y>amide or a co-amide, in which the amine component is a nitrogenous three-membered ring compound (aziridine). Alkali or alkali hydrogen sulphite can be added to both types of compounds, whereby compounds with the general formula (I) are formed.

In method variant g) the alkali salt of ct-aminoglutarimide or ct-aminosuccinimide is first formed and this is then alkylated on the imide nitrogen. As an alkylating agent, e.g. 2-bromoethanesulfonic acid or 3-bromopropanesulfonic acid is used. The N-sulfoethyl or N-sulfopropyl derivative obtained in this way is then partially hydrolysed in a weakly alkaline medium, whereby the u-amide is predominantly formed from the ring compound. This can be freed from the small amounts of α-amide by ion exchange chromatography.

Example 1

Preparation of γ-L-glutamyl taurine

(a) 40.85 g (0.11 mol) of carbobenzyloxy-L-glutamic acid α-benzyl ester (Liebig's Annals 655, 200, 1962) is dissolved in 500 ml of acetonitrile. The solution is cooled to -15° C. with the exclusion of humidity. While stirring, first 15.4 ml (0.11 mol) of triethylamine and then 15.4 ml (0.11 mol) of chloroformic acid isobutyl ester are added dropwise to the solution. The reaction mixture is stirred at -15° C. for 40 minutes, and then 28 ml (0.2 mol) of triethylamine is added, followed by 11.26 g (0.05 mol) of cystamine hydrochloride and finally 250 ml of acetonitrile. The mixture is stirred for another 2 hours

-15° C, and then at room temperature for a further 4 hours.

At the end of the reaction time, the mixture is evaporated

at 30° C. in 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. in vacuum. The residue is introduced together with 250 ml of water and 500 ml. ethyl acetate in a separatory funnel and the organic phase is separated. The organic phase is successively shaken with first 250 ml

water, then with 2 x 250 ml 5% sodium carbonate solution,

then with 2 x 250 ml IN hydrochloric acid and finally with 250 ml water.

(From the aqueous phase obtained by shaking with sodium carbonate solution, approximately 5 g of unreacted carbobenzyloxy-L-glutamic acid-α-benzyl ester can be recovered by acidifying with hydrochloric acid and shaking with ether). The ethyl acetate phase is dried over anhydrous sodium sulfate and then evaporated in vacuo at 30° C. to dryness. A thick, oily residue is obtained, which immediately solidifies

down to a crystalline mass. This is triturated 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. 2?.3 g of N,N'-bis-(N-carbobenzyloxy-γ-(a-benzyl)-L-glutamyl)-cystamine is obtained which melts at 91 - 92° C.

Elemental analysis for C44H5oN4°ioS2 ^M = 859 .05^

Calculated: 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% (b) 25.77 g (0.03 mol) of the N, N obtained in example 1(a) 1 -

bis-(N-carbobenzyloxy-γ-(α-benzyl)-L-glutamyl)-cystamine is dissolved in 75 ml of glacial acetic acid. A freshly prepared mixture of 75 ml of 30% hydrogen peroxide and 225 ml of glacial acetic acid is added dropwise over the course of 15 minutes to the ice-cooled solution. After addition, the cooling is removed and the reaction mixture is stirred at room temperature for 4 hours. It is then evaporated in a vacuum at 30°C. The oily product is first dried in a desiccator over phosphorus pentoxide and then over solid potassium hydroxide. 28.5 g of carbobenzyloxy-γ-(α-benzyl)-L-glutamyltaurine is obtained. The raw product can be used without purification for the production of γ-L-glutamyl taurine.

(c) 26.32 g (55 mmol) of the carbobenzyloxy-γ-(α-benzyl)-L-glutamyltaurine prepared in example 1(b) are dissolved in 50 ml of glacial acetic acid. 4 mol of hydrogen bromide dissolved in 50 ml of glacial acetic acid is added to the solution. A lively carbon dioxide development is observed. The reaction mixture is allowed to stand at room temperature for 2 hours

and then evaporated in vacuo at 30° C. The oily residue is dissolved in 170 ml of water and shaken out with 5 x 70 ml of ether. The water phase is evaporated in vacuo at 35° C. 20.42 g of γ-(α-benzyl)-L-glutamyltaurine is obtained, which is recrystallized from 90% ethanol.

Temp. 204 - 208°C.

Rf (n-butanol-pyridine-glacial acetic acid-water (15:10:3:12) = 0.53 R4 (n-butanol-glacial acetic acid-water 4:1:1) = 0.39 (d) 20.42 g of the γ-(α-benzyl)-L-glutamyltaurine obtained in example 1(c) is dissolved in 150 ml IN potassium hydroxide solution. The solution is left at room temperature for 4 hours and then applied to a Dowex® 50 x 2 (Fluka, 100 - 200 mesh) column 2 cm in diameter and 100 cm high. Elute there with water. 300 ml of eluate is collected from the beginning of the washout. This is evaporated in vacuum at 35° C. The oily residue is crystallized by adding 8 - 10 ml of water and about 100 ml of ethanol. After filtration, washing with alcohol and drying, 13.7 g of Y-L-glutamyl taurine is obtained. The crystalline product is recrystallized from 80% aqueous alcohol. 9.79 g of pure product is obtained, which corresponds to 70% calculated for N,N'-bis-(N-carbobenzyloxy-γ-(α-benzyl)-L-glutamyl)-cystamine.

The formed γ-L-glutamyltaurine has an IR spectrum which for the catacteristic groups shows the following maxima: •OHNH3+ x OH 3600-2400; NH (amide) 3315; CO (carboxyl) 1758; -) CO (amide) 1666 ; -5 NH3<+> 1576; ■) S03~ 1296, 1031 cm"<1.>

Example 2

Preparation of γ-L-glutamyl taurine

(a) 5.29 mg (1.1 mmol) of the carbobenzyloxy-γ-(α-benzyl)-L-glutamyltaurine prepared in example 1(b) is dissolved in 5 ml of potassium hydroxide solution and allowed to stand at room temperature for 4 hours. The solution is then shaken with 3 x 3 ml of ether. The water phase is applied to a Dowex 50x2 filled column (1 cm in diameter and 20 cm high) and eluted with water. 50 ml of solution is collected and evaporated in a vacuum at 35° C to dryness. Crude carbobenzyloxy-γ-L-glutamyl taurine is obtained, which is purified by paper electrophoresis at pH 6.5. The relative mobility in relation to the cysteic acid amounts to 1.05.

Rf (n-butanol-pyridine-glacial acetic acid-water 15:10:3:12) = 0.57

(b) The carbobenzyloxy-γ-L-glutamyl taurine prepared according to example 2(a) is dissolved in 2 ml of glacial acetic acid containing 4 mol of hydrogen bromide. The mixture is left at room temperature for 1/2 hour and then evaporated in a vacuum at 35°C. The residue is triturated several times with ether and then separated from the ether by decantation.

The crude product is recrystallized several times from 80% ether. 2.0 2 g of pure end product is obtained, which corresponds to a yield of 66% calculated for N,N'-bis-(N-carbobenzyloxy-y-(a-benzyl)-L-glutamyl)-cystamine. The pure product melted at 219 - 220° C. (a}20 = +14° (water, c = 1.02). The relative mobility in relation to cysteine acid with paper electrophoresis at pH 6.5 is 0.73, at pH 1.8 0.53.

R^ (n-butanol-pyridine-glacial acetic acid-water 15:10:3:12) = 0.19.

Analysis for C^^OgS (M = 254.27).:

Calculated: C 33.07%, H 5.55%, N 11.02%, O 37.75%, S 12.61% Found : C 33.15%, H 5.76%, N 10.94% , O 37.53%, S 12.17%

Example 3

Preparation of γ-L-glutamyl taurine

5.79 g (12.1 mmol) of the carbobenzyloxy-y<->(a-benzyl)-L-glutamyltaurine prepared in example 1(b) is dissolved in a mixture of 100 ml of ethanol and 25 ml of water and hydrogenated while shaking in the presence of 0.5 g of 10% palladium charcoal as catalyst. The catalyst is suitably added in two portions, each of 0.25 g. When no more hydrogen uptake occurs, the solution is filtered and then evaporated at 30° C in a vacuum. The oily residue is dried in a desiccator and phosphorus pentoxide. 3.1 g of γ<->L-glutamyl taurine is obtained, which is well soluble in water but not soluble in alcohol. By adding a little 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 end product is obtained, which corresponds to a yield of 66% calculated for N,N'-bis-[N-carbobenzyloxy-γ-(a-benzyl)-L-glutamyl]-cystamine. The pure product melted at 219 - 220°C. [a^° = +14° (water, c = 1.02). The relative mobility in relation to cysteic acid with paper electrophoresis at pH 6.5 is 0.73, at pH 1.8 0.53.

Rf (n-butanol-pyridine-glacial acetic acid-water 15:10:3:12) = 0.19.

Analysis for C^-^N^S (M = 254.27):

Calculated: 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%

The IR spectrum shows the following maxima for the characteristic groups: • J NH3+ x OH 3600-2400; -J NH (amide) 3315; s> CO (carboxyl) 1758 ; ■) CO (amide) 1666 ; ^NH3<+> 1576; •5S03~ 1296, 1031 cm"<1>.

Example 4

Preparation of γ-L-glutamyl taurine

5.42 g (11 mmol) of carbobenzyloxy-L-glutamic acid (α-benzyl)-γ-p-nitrophenyl ester (Chem. Ber. 9_6, 204, 1963) are dissolved in 50 ml of pyridine. The solution is cooled to 0° C. and a solution of 1.25 g (10 mmol) of taurine is added dropwise over the course of h hour with intense stirring. 3.08 ml (22 mmol) of triethylamine is then added to the mixture, and cooling and stirring are stopped. The mixture is left at room temperature for 72 hours and then evaporated in a vacuum. The residue is dissolved in 50 ml of water and 1N hydrochloric acid is added to the yellow solution until it is decoloured. To remove the p-nitro-phenol, the solution is shaken with 10 x 50 ml of ether. The water phase is evaporated in a vacuum. 6.9 g of carbobenzyloxy-Y-(α-benzyl)-L-glutamyltaurine triethylammonium salt are obtained.

The compound is catalytically hydrogenated as described in example 3, and the solvent is removed by evaporation in a vacuum. To remove triethylamine, the material is dissolved in a little water and applied to a Dowex<®> 50x2 column 2 cm in diameter and 40 cm high. Elute with water. One picks up approx. 120 ml of eluate is evaporated in vacuum at 35° C. Crystallization and isolation of the product takes place as described in example 3. 1.72 g of γ-L-glutamyltaurine is obtained, which corresponds to a 68% yield calculated for taurine.

The IR spectrum was as indicated in Example 3.

Example 5

(a) 0.48 g (1 mmol) of carbobenzyloxy-ct-L-glutamyl-(γ-p-notrophenyl ester)-glycine ethyl ester (Acta Chim. Acad. Sei. Hung. 65, 375 , 1890) is dissolved in 6 ml of ethyl acetate. The solution is cooled with ice water. At 0° C, first add 0.08 g of the solution

(1 mmol) of cysteamine in 1 ml of dimethylformamide, and then 0.14 ml (1 mmol) of triethylamine is added dropwise. Slowly, a precipitate begins to precipitate. The reaction mixture is kept for some time in ice water, then left for one day at room temperature and finally diluted with a 1:1 mixture of ether and ethyl acetate. The precipitate is centrifuged off and washed first with a 4:1 mixture of ether and ethyl acetate and then several times with ether. After drying over sulfuric acid, the precipitate is washed three times with IN hydrochloric acid.

twice with water, twice with saturated sodium hydrogen carbonate solution and finally again twice with water, and then dried in vacuum over sulfuric acid. 0.35 g of carbobenzyloxy-α-L-glutamyl-(γ-cysteamine)-glycine ethyl ester is obtained, which corresponds to a yield of 85%.

Analysis for Cl8<H>25<06N>3<S> (M = 411.4):

Calculated: C 52.6%, H 6.13%, S 7.8%

Found : C 53.4 i, H 6.5 i, S 7.7 8

The infrared spectrum shows the following maxima for the characteristic groups: NH 3310 cm"<1>, C=0 (COOEt) 1748 cm"<1>, C=0 (Z) 1690 cm-1, C=0 (amide) 1655 cm"<1>.

Dissolve 100 mg of carbobenzyloxy-α-L-glutamyl-(γ-cysteamine)-glycine 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 allowed to stand for 4 hours under ice water cooling. The course of the reaction is followed by paper electrophoresis. After dilution with water, the reaction mixture is lyophilized. You get the final product in the form of a foam. 0.11 g of carbobenzyloxy-α-L-glutamyl-(γ-taurine)-glycine ethyl ester (95%) is obtained.

b) 100 mg of the carbobenzyloxy-α-L-glutamyl-(γ-taurine)-glycine ethyl ester prepared according to example 5(a) is dissolved in a

mixture of 1 ml of trifluoroacetic acid and 1 ml of concentrated hydrochloric acid. The solution is kept for 3 hours at 35° C in a closed firing tube. The solution is then evaporated in vacuo, the residue is digested with ether and n-hexane and then evaporated again. A white amorphous substance is obtained which on electrophoretic examination proves to be uniform and gives a positive ninhydrin stain. The product is α-L-glutamyl-(γ-taurine)-glycine. Yield is 0.06 g (88%).

Analysis for C9<H>3<0>7S (M=311.3)

Calculated: S 10.3%

Found : S 10.0%

The infrared spectrum shows the following maxima for the characteristic groups: NH* 3100 cm"<1> (broad); OH (COOH)

3200 cm<_1> (wide); C=0 (COOH) 1730 cm"1; C=0 (amide-I) 1680 cm<-1>, C=0 (amide-II) 1560; S=0 (SO2OH) 122o cm"<1> (intensive );

S=0 (S020~) 1045 cm"<1> (intensive).

Hydrolysis: 20 mg of the compound is kept in 1 ml of 6N hydrochloric acid i

a fused glass tube for 24 hours at 105° C. After cooling, a sample of the solution is subjected to electrophoresis at pH 1.8. The solution contains glutamic acid, glycine and taurine. (c) 100 mg of the α-L-glutamyl-(γ-taurine) glycine prepared in example 5(b) is dissolved in 25 ml of a 0.2 molar ammonium bicarbonate buffer with pH 8.5. 1 mg of carboxypeptidase A dissolved in 0.5 ml of water is added to the solution. (Producer of the enzyme: Serva, Heidelberg). The mixture is kept at 37° C in a thermostat for 24 hours and then lyophilized. Y~L-glutamyl taurine and glycine can be detected in the dry lyophilisate. The pure γ-glutamyl-taurine can be recovered by electrophoresis or chromatography on a Dowex 50 column.

Example 6

Preparation of γ-L-glutamyl taurine

(a) 0.47 g (1 mmol) of carbobenzyloxy-α-L-glutamyl-(γ-p-nitrophenyl ester) glycine methyl ester is dissolved in 6 ml of pyridine. Under ice-cooling, first add 0.125 g (1 mmol) taurine in 2 ml water, and then 0.28 ml (2 mmol) triethylamine in such small portions that the solution always remains clear. The reaction mixture is left at room temperature for 3 days. After evaporation in a vacuum, an oil is obtained which, after digestion in ether and then in petroleum ether, is passed in a vacuum over sulfuric acid. Carbobenzyloxy-α-L-glutamyl-(γ-taurine)-glycine methyl ester is obtained. (b) 100 mg of the carbobenzyloxy-α-L-glutamyl-(γ-taurine)-glycine methyl ester prepared according to example 6(a) is reacted with 4.ml of 2N acetic acid hydrogen bromide at room temperature, until all the substance has dissolved (approx. 30 my). The clear solution obtained is poured into 30 ml of ether and left in a cool place for 1 day. The formed precipitate is centrifuged off, washed several times with ether and then dried in a vacuum over potassium hydroxide, sulfuric acid and phosphorus pentoxide. As the electrophoretic examination shows, the hydrogen bromide of the α-L-glutamyl-(γ-taurine) glycine methyl ester is obtained in practically pure form. (c) The salt obtained according to example 6(b) is reacted under ice water cooling for 3 hours with 2 ml IN sodium hydroxide solution. The course of the hydrolysis is followed by electrophoresis. The reaction mixture is subjected to an ion exchange with 10 ml of Dowex 50 in the H form and then lyophilized. As the electrophoresis still shows impurities, the product is recrystallized several times from aqueous ethanol to obtain the corresponding purity. 40 mg (59 "%) of α-L-glutamyl-(γ-taurine)-glycine is obtained.

The infrared spectrum shows the following maxima for the characteristic groups: NH 3310-1, NH3+ 3100 cm"1 (broad), C=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).

(d) 100 mg of the ct-L-glutamyl-(Y-taurine) glycine prepared in example 6 (c) is dissolved in 25 ml of a 0.2 molar ammonium bicarbonate buffer with pH 8.5. 1 mg of carboxypeptidase A dissolved in 0.5 ml of water is added to the solution. (Producer of the enzyme: Serva, Heidelberg). The mixture is kept at 37°C in a thermostat for 24 hours and then lyophilized. Y-L-glutamyl taurine and glycine can be detected in the dry lyophilisate. The pure γ-L-glutamyl-taurine can be recovered by electrophoresis or chromatography on a Dowex 50 column.

The IR spectrum shows the following maxima for the characteristic groups: NH^ x OH 3600-2400; ^ NH (amide) 3315;

CO (carboxyl) 1758 ; >> CO (amide) 1666 ; -3 NH_,+ 1576 ; JS0~ 1296 ,

-1 J 3

1031 cm x.

Example 7

Preparation of γ-L-glutamine homotaurine

(a) 1.08 3 g (2.2 mmol) of carbobenzyloxy-L-glutamic acid-(α-benzyl)-γ-p-nitrophenyl ester are dissolved in 6 ml of a mixture of pyridine and water in the ratio 2:1. First 278 mg (2 mmol) homotaurine and then 0.59 ml (4.2 mmol) triethylamine are added to the solution. The yellow solution is prepared at room temperature

<

leave for 72 hours and then evaporate in a vacuum. The oily residue is dissolved in water, neutralized with hydrochloric acid and then extracted with ether for 8 hours in a continuous extractor to remove the p-nitrophenol. The water phase is evaporated in a vacuum. 1.68 g of carbobenzyloxy-γ-(α-benzyl)-L-glutamyl homotaurine is obtained.

(b) The entire quantity of the material prepared according to example 7(a) (1.68 g) is dissolved in 10 ml of 50% aqueous ethanol, added

0.3 g of 10% palladium activated carbon and hydrogen are then passed through the suspension for 4 hours. The solution is then filtered and evaporated in a vacuum. The residue is dissolved in 1 - 2 ml of water and passed over a Dowex 50 x 2 (H shape) column with a diameter of 1 cm and a height of 35 cm. Elute with water. 50 ml of eluate is collected and then evaporated in vacuum. 440 ml of γ-L-glutamyl homotaurine is obtained as a residue, which corresponds to a yield of 82%. The electrophoresis carried out at pH 6.5 shows a slight contamination of partially neutral, partially acidic substances (homotaurine or glutamic acid). The product can be cleaned e.g. by preparative electrophoresis.

Both in the electrophoresis at pH 6.5 and in the electrophoresis carried out at pH 1.8, the compound migrates in the direction of the cathode. Its relative mobility in relation to .cysteine acid constitutes

at pH 6.5 0.68, and at pH 1.8 0.50.

Rf (n-butanol-pyridine-glacial acetic acid-water 15:10:3:12) = 0.19.

The γ-L-glutamyl homotaurine formed gives an IR spectrum

which for the characteristic groups shows the following maxima:

■) -NH = 3345; -0 NH3+, OH = 3200-2200, \) CO = 1740;

•OCO-NH = 1645; >) S03~ = 1240, 1190, 1150, 1038, J-NH = 1570, 1535 >)S03~ = 590 cm"<1>

Example 8

Preparation of γ-L-glutamyl-N-methyltaurine

(a) 1.083 g (2.2 mmol) of carbobenzyloxy-L-glutamic acid-(α-benzyl-γ-p-nitrophenyl ester) are reacted in the manner described in example 7 with 278 mg (2 mmol) of N-methyltaurine. 1.59 g are obtained carbobenzyloxy-γ-(a-benzyl)-L-glutamyl-N-methyltaurine (b) The entire quantity of the material prepared according to example 8(a) (1.59 g) is hydrated in the manner described in example 7(b) 4 23 mg of γ-L-glutamyl-N-methyltaurine is obtained, which corresponds to a yield of 79%.

The compound migrates both at pH 6.5 and at pH 1.8

in paper electrophoresis in the direction of the anode. The relative mobility in relation to cysteic acid: pH 6.5: 0.68; pH 1.8: 0.49.

Rf (N-butanol-pyridine-glacial acetic acid-water 15:10:3:12) = 0.16.

Example 9

Preparation of γ-L-glutamyl-L-cysteine acid

(a) 2.87 g (6.6 mmol) of carbobenzyloxy-L-glutamic acid-(a-benzyl)-γ-p-nitrophenyl ester are dissolved in 20 ml of pyridine and a solution of 1.25 g (6 mmol) of L-cysteine monohydrate in a mixture of 17 ml of water and 17 ml of pyridine. After adding 2.6 ml (18.6 mmol) of triethylamine, the reaction mixture is allowed to stand at room temperature for 72 hours. The solution is then evaporated in vacuo at 30° C. The residue is dissolved in 20 ml of water, made acidic with concentrated hydrochloric acid and then shaken out with 15 x 10 ml of ether. The water phase is evaporated in vacuo at 35° C. Carbobenzyloxy-γ-(α-benzyl)-L-glutamyl-L-cysteine acid is obtained. (b) The product prepared in example 9(a) is dissolved in 20 ml of water, 0.3 g of 10% <p>alladium activated carbon is added, and hydrogen is passed through the suspension for 3 hours. The reaction mixture is worked up in the manner described in example 7(b). γ-L-glutamyl-L-cysteine acid is obtained which melts at 187°C. The relative mobility in relation to cysteic acid by paper chromatography:

at pH 6.5: L21; at pH 1.8: 0.54.

The formed γ-L-glutamyl-L-cysteine acid has an IR spectrum which for the characteristic groups shows the following maxima:

■0 NH (amide) 3423; ^ NH3+' OH 3300-2200 OCO (carboxyl) 1748, 1712; 0 CO (amide) 1627; -)NH (amide) 1527; 0 SO ~ 1221, 1109, 1043; S03" 525cm"<1>

Example 10

Preparation of γ-L-glutamylcholamine phosphate

(a) 1.083 g (2.2 mmol) of carbobenzyloxy-L-glutamic acid-(α-benzyl)-γ-p-nitrophenyl ester is dissolved in 6 ml of a mixture of pyridine and water in the ratio 2:1. 2.8 mg (2 mmol) cholamine phosphate (US patent no. 2 730 542) is then added to the solution and

then 0.87 ml (6.2 mmol) of triethylamine. The reaction mixture is left at room temperature for 72 hours and then evaporated in vacuo. The further processing takes place in the manner described in connection with carbobenzyloxy-γ-(α-benzyl)-L-glutamylhomotaurine (Example 7). 1.25 g of carbenzyloxy-γ-(α-benzyl)-L-glutamylcholamine phosphate is obtained. (b) The entire amount of the product recovered in Example 10(a) (1.25 g) is subjected to catalytic hydrogenation to remove the protecting groups. The hydrogenation as well as the purification by ion exchange is carried out in the manner described in connection with the preparation of γ-L-glutamyl homotaurine (Example 24). 470 mg of γ-L-glutamylcholamine phosphate is obtained, which corresponds to a yield of 91%. However, as evidenced by paper electrophoresis, the product contains as contamination approx. 15 - 20% cholamine phosphate. Among other things, electrophoresis is used as a purification method.

In paper electrophoresis, the compound migrates both at pH 6.5 and at pH 1.8 in the direction of the anode. The relative mobility in relation to cysteic acid: pH 6.5: 0.75; pH 1.8: 0.36.

Rf (n-butanol-pyridine-glacial acetic acid-water 15:10:3:12) = 0.18.

Example 11

Preparation of @-L- aspartyl taurine

(a) 526 mg (1.1 mmol) of carbobenzyloxy-L-aspartic acid-(α-benzyl-6-p-nitrophenyl ester (Chem. Ber. 9_7, 1789, 1964)) are dissolved in 5 ml of pyridine. The solution is cooled to 0 ° C and then a solution of 125 mg (1 mmol) taurine in 2 ml water and then 0.28 ml (2 mmol) triethylamine is added in small portions. The reaction mixture is left at room temperature for 48 hours and then evaporated in vacuo. The residue is dissolved in 5 ml of water, and to the initially yellow solution, 1N hydrochloric acid is added until it is decoloured. To remove the p-nitrophenol, the solution is shaken with 10 x 5 ml of ether. The water phase is evaporated in vacuo. 478 g of carbobenzyloxy-S-(a -benzyl)-L-aspartyl taurine. (b) The entire quantity of the product obtained in example 11(a) is dissolved in 6 ml of 50% aqueous ethanol. 100 mg of 10% palladium activated carbon is added to the solution and then hydrogenated at 4 h er's passage of hydrogen gas. After filtration and evaporation in a vacuum, the triethylamine on it is removed in connection with the production of γ-L-glutam yl homotaurine (Example 7(b)) described manner by ion exchange. 172 mg of p-L-aspartyl taurine is obtained, which corresponds to a yield of 71%. The product is contaminated with taurine, which can, among other things, be removed by electrophoresis.

In paper electrophoresis, the compound migrates to the anode both at pH 6.5 and at pH 1.8. The relative mobility in relation to cysteic acid: pH 6.5: 0.77; pH 1.8: 0.58.

Rf (n-butanol-pyridine-glacial acetic acid-water 15:10:3:12) = 0.16

Example 12

Preparation of p-L-aspartyl homotaurine

(a) 526 mg (1.1 mmol) carbobenzyloxy-L-aspartic acid-(α-benzyl)-β-p-nitrophenyl ester and 1.39 g (1 mmol) homotaurine are reacted in the manner described in example 11(a). Carbobenzyloxy-Ø-(α-benzyl)-L-aspartyl homotaurine is obtained. (b) The product obtained in example 12(a) is catalytically hydrogenated in the manner described in example 7(b). 203 mg (84%) [3-L-aspartyl homotaurine is obtained.

In paper electrophoresis, the compound migrates both at pH 6.5 and at pH 1.8 in the direction of the anode. The relative mobility in relation to cysteic acid: pH 6.5: 0.72; pH 1.8: 0.53.

Rf (n-butanol-pyridine-glacial acetic acid-water 15:10:3:12) = 0.17.

Example 13

Preparation of 3-L-aspartylcholamine phosphate

(a) Carbobenzyloxy-L-aspartic acid (α-benzyl)-3_p-nitro-phenyl ester and cholamine phosphate are reacted in the manner described in example 10(a). Carbobenzyloxy-g-(α-benzyl)-L-aspartylcholamine phosphate is obtained. (b) The substance obtained in example 13 (a) is catalytically hydrogenated in the manner described in example 7(b). You get (3-L-aspartylcholamine phosphate.

In paper electrophoresis, the compound migrates both at pH 6.5 and at pH 1.8 in the direction of the anode. The relative mobility in relation to cysteic acid: pH 6.5: 0.81; pH 1.8: 0.40.

Rf (n-butanol-pyridine-glacial acetic acid-water 15:10:3:12) = 0.14.

Example 14

Y-L-glutamine taurine (prepared according to the above examples, can be purified by recrystallization in the following way: 300 mg of crude compound is dissolved at room temperature with stirring in 5 ml of anhydrous dimethylsulfoxide. The opalescent solution is filtered and the filter is washed out with 0.5 ml dimethylsulfoxide. The filtrate is combined with the washing liquid and 55 ml of absolute ethanol is added. The mixture is left at room temperature for 12 hours, then the compound is filtered off, it is washed with 2.5 ml of absolute ethanol and dried in a vacuum desiccator over phosphorus pentoxide to constant weight. You get 240 mg crystalline Y-L-glutamyl taurine (yield of the recrystallization: 80%).

Instead of ethanol, the same amounts of dioxane, ether or acetone can be used for the recrystallization. The quality of the crystalline product is the same in all cases. Melting point (according to Boetius): 218 - 219°C. The product is layer chromatographically uniform.

Example 15

In the manner suitable for the preparation of glutamic acid γ-amides (Acta Chim. Acad. Sei. Hung. 6_4, 285, 1970) carbobenzyloxy-γ-(α-benzyl)-L-glutamylcholamine is prepared. 4.14 g (14 mmol) of this product are dissolved in 50 ml of absolute pyridine and 9 g (33 mmol) of diphenyl phosphoric acid chloride are added. The reaction mixture is kept at 0° C for 12 hours and then diluted with 80 ml of chloroform . The separated product is filtered off, washed with dilute hydrochloric acid and then with water, and finally dried in a desiccator over potassium hydroxide. The dry compound dissolves

in 15 ml glacial acetic acid containing 3.3 mol/l hydrogen bromide, the solution is allowed to stand for 15 minutes and then evaporated in vacuo at

35° C. The residue is dissolved in 30 ml IN sodium hydroxide solution and the solution is left for 1 hour at room temperature. Its pH value is then set to 4 with acetic acid, and to remove phenol and benzyl alcohol, the solution is extracted with 3 x 30 ml of ether. The water phase is applied to a column with Dowex 50 in the H form, and eluted with water. The eluate is evaporated in a vacuum and the residue is recrystallized from a mixture of acetone and water in the ratio 2:1. 0.8 g of γ-L-glutamylcholamine phosphate is obtained.

Example 16

4.68 ml (5 mmol) of phosphorus oxychloride are added dropwise while cooling with stirring in 1.8 ml of water (Biochem. Preparations 6_,

76, 1958). To the solution is added in small portions 1.9 g (10 mmol) γ-L-glutamylcholainin (prepared from carbobenzyloxy-Y-(ct-benzyl)-L-glutamylcholamine according to example 15). The mixture is stirred at 60° C for 2 hours, after cooling while stirring, 0.7 2 ml of water is added and then allowed to stand at room temperature for 2 hours. 10 ml of 96% ethyl alcohol and then 10 ml of ether are then added dropwise while stirring. The reaction mixture is left at 4° C overnight, and then 5 ml of 96% ethanol is added.

The precipitated compound is filtered off, washed first with ethanol and then with ether, and finally recrystallized from a mixture of ethanol and water. 1.75 g of γ-L-glutamylcholamine phosphate is obtained.

Example 17

4.14 g (10 mmol) of carbobenzyloxy-^-(α-benzyl)-L-glutamylcholamine are dissolved in 40 ml of pyridine and the solution is cooled to -10° C. In small portions, with intense stirring, 2.1 g (11 mmol) p-toluenesulfonyl chloride. The reaction mixture is stirred at 0° C. for 3 hours and then poured onto 40 g of melting ice. The separated precipitate is filtered off, washed with water and finally recrystallized from a mixture of ethanol and petroleum ether. The product is subjected to catalytic hydrogenation in the manner described in example 5. The dried substance obtained after hydrogenation is dissolved in 30 ml of water, and 10.1 g is added to the solution

(40 mmol) sodium sulfite heptahydrate. The solution is stirred at 40° C. for 24 hours and then evaporated in vacuo. The residue is dissolved in a little water and applied to a column of Dowex 50, from which it is eluted with water. The eluate is evaporated in vacuo and the residue is dried over potassium hydroxide. After recrystallization from 80% ethanol, 1.6 g of γ-L-glutamyltaurine is obtained.

Example 18

15 ml of thionyl bromide is added to 4.14 g (10 mmol) of carbobenzyloxy-γ-(α-benzyl)-L-glutamylcholamine, and the mixture is stirred for 3 hours. Ether is then added, the precipitate is filtered off and recrystallized from a mixture of acetone and petroleum ether. The compound obtained is dissolved in a mixture of dimethylformamide and water, and 10.1 g (40 mmol) of sodium sulphite heptahydrate are added to the solution while stirring. The mixture is stirred for 24 hours at room temperature and then for a further 6 hours at 50° C, and finally filtered. The clear solution is evaporated in a vacuum, and the residue is catalytically hydrogenated in the manner described in example 5. The dried compound obtained after hydrogenation is dissolved in a little water, applied to a column with Dowex ® 50 and eluted with water. The eluate is evaporated in vacuo and the residue is recrystallized from 80% ethanol. 1.2 g of Y-L-glutamyl taurine is obtained.

Example 19

3.71 g (10 mmol) of carbobenzyloxy-L-glutamic acid α-benzyl ester are dissolved in 60 ml of acetonitrile. The solution is cooled to -15° C and, with stirring, first 1.4 ml (10 mmol) of chloroformate isobutyl ester and then 1.4 ml (10 mmol) of triethylamine are added. The reaction mixture is stirred for 30 minutes at -15° C and then 2.05 g (10 mmol) of bromethylamine hydrogen bromide, 1.4 ml (10 mmol of triethylamine and 40 ml of acetonitrile cooled to -15° C) are added. The mixture is stirred at -15° C for 2 hours and then for another 4 hours at room temperature. The filtrate is then filtered and the filtrate is evaporated in a vacuum at 35° C. The residue is dissolved in a mixture of dimethylformamide and water, and 10.1 g of sodium sulphite heptahydrate is added to the solution. After working up the mixture in the manner described in example 18, 1.45 g of γ-L-glutamyl-taurine is obtained.

Example 20

3.02 g (10 mmol) of carbobenzyloxy-L-glutamine sodium salt (Liebigs Annalen 640, 145, 1961) are dissolved in 50 ml of dimethylformamide. The solution is added to a 12 mmol sodium hydride containing oily suspension, and then heated for 2 hours while excluding atmospheric moisture. The solution of 2.11 g (10 mmol) of sodium bromoethanesulphonic acid in 50 ml of dimethylformamide is then added dropwise and the mixture is further heated for 2 hours. It is then evaporated in vacuo, the residue is extracted with ether and then dried. The dry compound is dissolved in water, brought onto a column

with Dowex<®> 50 and diluted with water. The eluate is evaporated in vacuo and the residue is dried over potassium hydroxide. The dried compound is catalytically hydrogenated in the manner described in example 3.

After recrystallization from ethanol/water, 1.55 g of γ-L-glutamyl taurine is obtained.

Example 21

2.58 g (10 mmol) of L-N-(2,6-dioxo-3-piperidyl)-phthalimide (J. Pharm. Sei. 5J7, 757, 1968) are dissolved in a sodium ethylate solution prepared with absolute ethanol. The solution is evaporated in vacuo, the residue is dissolved in 50 ml of dimethylformamide and 2.25 g (11 mmol) of sodium bromomethanesulphonic acid is added to the solution while stirring. The mixture is heated for 2 hours and then evaporated in vacuo. The residue is dissolved in water, placed on

a column with Dowex ® 50 and eluted with water. The eluate is evaporated in a vacuum and the residue obtained is boiled for 3 hours in 100 ml of 0.5N hydrochloric acid. After evaporation of the solution, 30 ml of ethanol and 0.7 ml of 72% hydrazine hydrate are added to the material, and the mixture is boiled for 1 hour. The mixture is then evaporated in vacuo. The residue is taken up in 25 ml of 2N hydrochloric acid and the mixture is maintained

50° C for 10 minutes and then set aside at room temperature for 30 minutes. The separated solid is removed by filtration, the filtrate is evaporated in vacuo and the residue is dried in a desiccator over potassium hydroxide. The dried substance is dissolved in a mixture of water, acetic acid and formic acid in the volume ratio 90:8:2, and the solution is applied to a column of Dowex ® 1 with a diameter of 2 cm and a height of 100 cm. The column is first brought into equilibrium with the aforementioned solvent mixture. First elute with the aforementioned solvent mixture, and 400 ml of eluate is collected. This fraction contains Y~L-glutamyl taurine. It is then eluted with 0.5N hydrochloric acid, with 400 ml of eluate also being collected. This fraction is evaporated to dryness in vacuum at 35° C. The residue is dried in a desiccator over solid sodium hydroxide and then recrystallized from 80% ethanol. 1.07 g of Y~L-glutamyl taurine is obtained.

Example 22

4.43 g (10 mmol) carbobenzyloxy-L-pyroglutamic acid-dicyclohexylamine salt (Liebigs Annalen 640, 145, 1961), 1.25 g (10 mmol) taurine and 0.84 g (10 mmol) sodium bicarbonate are dissolved in 50 ml of water. The solution is heated for 4 hours (or left at room temperature for 24 hours) and then evaporated in vacuo. The residue is dissolved in water, applied to a column with

Dowex ®50 and eluted with water. The eluate is evaporated. The compound obtained is catalytically hydrogenated in the manner described in example 3. After recrystallization from an ethanol-water mixture, 2.03 g of γ-L-glutamyltaurine is obtained.

Example 23

7.38 g (15 mmol) carbobenzyloxy-L-glutamic acid (α-benzyl)-Y-p-nitrophenyl ester and 1.41 g (10 mmol) cholamine sulfate

(P. A. Leighton: Am. Chem. Soc. 6_9, 1540, 1947) are reacted with each other in the manner described in example 7(a). The preparation of the reaction mixture, the removal of the protective group by catalytic hydrogenation and purification of the product takes place in the manner described in example 3. 1.75 g of Y-L-glutamyl-cholamine sulfate (64.7%) is obtained. Melting point: 164-166°C.

The infrared spectrum shows for the characteristic groups the following maxima: Y-NH- = 3365, yNH3 + OH = 3200-2400;

yCO = 1748, yCO-NH = 1660; YS03~ = 1260, 1186, 1155,

989, 829,

-NH- = 1565, 1540

S03~ = 580, 595 cm"<1>.

Claims (1)

Analogous procedure for the preparation of new therapeutically active amino acid derivatives with the general formula: where R is hydrogen or alkyl with 1-4 carbon atoms, R^ is hydrogen or carboxyl, B<1> is -SH, -SO 2 OH, -SO OH or -OPO(OH) 2 , n is an integer from 1 to 4, m is an integer from 1 to 3, and t is an integer from 1 to 3, and their pharmacologically acceptable salts and optically active isomers, characterized in that one a) in compounds with the general formula: 5 1 7 where R, R , B , n, m and t are as indicated above, and R is alkoxycarbonyl with 1-4 carbon atoms, or aralkylcarbonyl with 7-9 carbon atoms which is optionally substituted with one or more of the substituents halogen, alkoxy or nitro, removes the protecting group R^ by acidolysis, hydrolysis, or catalytic hydrogenation, or b) subject to a compound of the general formula: where R, R 5 , B 1 , n, m and t are as indicated above, and A 2 is (1) alkoxy with 1-4 carbon atoms, or (2) aralkyl with 7-9 carbon atoms which may optionally be in the p-position substituted with alkoxy or nitro, or (3) the group (-NH-CH-CO) -Y- where R<6> is R<6 >hydrogen or alkyl with 1-4 carbon atoms, Y is hydroxy or alkoxy with 1-4 carbon atoms, and r is an integer from 1 to 10, an alkaline or enzymatic hydrolysis, an acidolysis or hydrogenolysis, or c) in a compound of the general formula: 5 17 3 where R, R B , R , n, m and t are as stated above, and A is (1) alkoxy with 1-4 carbon atoms or (2) an optionally in the p-position with alkoxy or nitro substituted aralkyl group with 7-9 carbon-7 3 atoms, removes the protecting groups R and A simultaneously by acidolysis, alkaline hydrolysis or hydrogenolysis, or d) submits a compound of the general formula: where R, R5, n, m and t are as stated above, and A is (1) hydroxy, (2) alkoxy with 1-4 carbon atoms, (3) an optionally substituted aralkyl group with 7 -9 carbon atoms, or (4) the group (-NH-CH-CO) -Y where R , r and Y are as indicated above under (b3); R6 r R1 is (1) hydrogen, (2) alkoxycarbonyl with 1-4 carbon atoms or (3) aralkylcarbonyl with 7-9 carbon atoms optionally substituted with one or more of the substituents halogen, alkoxy or nitro; and B 2 is -SH, -SOOH, -OH, p-toluenesulfonyloxy, halogen or -SC^R^O where R<10> is alkoxy with 1-4 carbon atoms, or aralkyl; oxidation or hydrolysis, reacts with alkali or alkali hydrogen sulphite, esterifies with sulfuric or phosphoric acid or a derivative thereof; and then optionally removes the protective groups attached to the u>-amino- and/or u-carboxyl groups, or e) for the preparation of compounds of formula I where B1 is a sulfonyl-containing group, reacts a compound with the general formula: 11 12 where R , A and n are as indicated above, R is hydrogen or alkyl with 1-4 carbon atoms, and R 1^ is an alkali metal, with alkali metal salts of haloalkylsulfonic acids, and then optionally remove protective groups attached to the io-amino- and/or o>-carboxyl groups; or f) for the preparation of compounds of formula I, where B<1> is -SO2OH, oxidizes a compound with the general formula: where R, R 5 , R 7 , n, m and t are as indicated above, and A 4 is hydroxy, aralkyloxy, preferably benzyloxy, substituted aralkyloxy, preferably p-methoxybenzyloxy or p-nitrobenzyloxy, and then 7 removes 4 the protecting groups R and A', or g) for the preparation of compounds of formula I where B<1> is -SO 2 OH, hydrolyze a compound of the formula: 15 1 2 where R , R , B , n, m and t are as indicated above ; and R is hydrogen, or R 1 and R 2 together with the nitrogen atom to which they are attached form a phthalimido group, or h) for the preparation of compounds of formula I where B 1 is -SC^OH, reacts a compound of the general formula : 7 4 7 where R, A and n are as indicated above, and A is hydroxy, p-nitro-phenyloxy, pentachlorophenyloxy or alkoxycarbonyloxy with 2-4 carbon atoms, with a compound of the general formula: where R, R~*, m and t are as indicated above, oxidise the obtained compound of the general formula (VII) and remove the protecting groups R^ and * or i) react a compound of the general formula (XVIII) with a compound of the general formula: where R, R 5 , B 1, m and t are as above, and removes protect-7 4 the other groups R and A', or j) in compounds with the general formula: 5 7 14 where R, R , R , B , A , n, m and t are as indicated above, in arbitrary order, the protecting group R 7 splits off by acidolysis and the protecting group A 4 by alkaline hydrolysis, and optionally transfer the obtained compounds to their pharmacologically acceptable salts or liberate them from their salts.