SI7512324A8 - Process for the preparation of dipeptids derivatives - Google Patents

Description

C '07C 103/52. A.6IK S7 / 02 by the e-halide of this acid, wherein in the formula 'Z is a group of the formula

CH,

I:

0, ^ N ^XX GQ - NH - CH

-GG - N and their · salts of nonactor histidylprolinamide. with the carboxylic acid of the formula

s.

R,

II

L σασΗ.

about ca.

At approx

O>

N l ^:

I *

R4

I —NH — CO— iii —C — S—

I

Rs gde- su R to R _by isti- iii Various i eznaeavaju · hydrogen iii alkyl grupu- self 1-3 GT-atom pričemu- Ki Ri i- uzett mjedna may form ^{- yes;} Cade an additional bond between the C-aitoms · to which they are attached. The one obtained by the process according to the invention · show a pronounced pmhostrmulative, ie. anti-depressant effect in humans' and the Holy One, and are stable in foaming with similar known compounds, especially in response to the hormone releasing thyrotropin ("Tyrotropin-Releasing-Hormone" - TRH).

- 39 330

The invention relates to a process for the preparation of dipeptide derivatives derived from histidylprolin, wherein one or both of the amino acids in this dipeptide may be optically active or racemic, especially in the form of an L-configuration. Novel dipeptide derivatives that are subjected to the process of the invention show valuable biological properties and can accordingly be used as medicaments.

The invention solves the problem of obtaining, in a simple manner, a suitable choice of starting material, new dipeptide derivatives with the desired better biological properties, especially in terms of quality and duration of action.

Pyroglutamyl-histidyl-prolinamide, known as "Tyrotropin-Releasing-Hormone" or "TRH" is naturally occurring in small quantities but can also be synthetically produced. It has a pronounced endocrinological and pharmacological effect, but it decays rapidly in the body so that it is practically impossible to use for therapeutic purposes. From the Chemiker-Zeitung 98, p. 554-561, in particular p. 556- ^ 557, it is seen that scientists dealing with this enclosure thought that a stable substitution for TRH was practically impossible to obtain because any substitution of pyroglutamyl residues in TRH would lead to a loss of its biological activity.

Now, by the process of the invention, new dipeptide derivatives of the general formula (I) are obtained:

/ ^R 1 ^x co

and their salts with. pharmaceutically acceptable acids, wherein R 1 and R 2 may be the same or different, and denote hydrogen atoms, alkyl groups of 1-3 C atoms, or together represent one additional bond between the two C atoms to which they are attached. R ₃ denotes a hydrogen atom or an alkyl group with

1-3 C atoms, Z denotes a group suitable for the faces of the outer 5- or 6-membered ring, e.g. NH Group III Formula Group:

OH R »

I I —N = C—, ie, —NH — CO—, —C — S— iii l • Rs

I —c — o—, I

R 5 where R ₄ and R _{5 are the} same or different and denote a hydrogen or an alkyl group having 1 to 3 C atoms. Usually the carboxylic acid of formula (I)

R.

/ ^R 1

II ^X COOH n

which is related to dipeptide, orotic acid, or thiomorpholine- (5) -one- (3) -carboxylic acid.

Other suitable acids of general formula (II) are, for example, thiomorpholine- (5) -one- (2,2) -dimethyl- (3) -carboxylic acid, 4-carboxy-imidazol-2-one, thiomorpholine- ( 6) -methyl- (5) ron- (3) -carboxylic acid, 5-methyl-, 5-ethyl- or 5-propyl-orotic acid, and other similar compounds.

When R! together with R ₂ does not form a bond between the carbon atoms to which they are attached and / or when R ₄ and R _{5 are} different from each other, then the acid residues of formula (II) may also be in racemic or optically active form, usually in the L-form, in the compounds of formula I.

Particularly represented representatives of the compounds of formula I are orotyl-histidyl-prolinamide and its salts, in which, above all, both amino acids become L-shaped.

Based on the basicity of the histidine residue, the compounds of formula I are capable of forming salts with acids. It is therefore an object of the present invention to provide salts of these compounds with pharmaceutically (in the form of salts) applicable inorganic or organic acids; such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoic acid, formic acid, acetic acid, propionic acid, benzoic acid, phenylacetic acid, benzolsulfonic acids and. t. d.

The compounds obtainable according to the invention exhibit biological properties which, in terms of quality of action, surprisingly and almost completely correspond to those of pyroglutamyl-histidyl-prolinamide, commonly referred to as "Tyrotropin-Releasing-Hoimone" or "TRH", but which nevertheless significantly longer than the properties of a known product. It is also of therapeutic importance that, in the compound of the invention, the ratio of centrally induced activity to endocrinological effects, compared to the known product (TRH), has shifted in favor of fanmacologically valuable properties. The compounds act in parenteral and oral administration, with the onset of rapid action; soil example at parenteral dose after about 15 minutes,

The symptoms that prevail are answered in the pharmacological test by central excitation. If the test animals were given equal doses of TRH, or orotyl-L-histidyl-L-propylamide, the product obtained by the process of the invention was shown to be five times more potent and several times longer than TRH.

The toxicity of the compound is negligible, so that it can be used, for example, as psycho-stimulants or antidepressants. Products can find a twinge in both humans and animals.

Suitable, pharmacologically applicable, formulations are tablets, dragees, granules, capsules, drops, juices, or syrups and sprays for intranasal use or for administration of substances via the bronchi, as well as aqueous sterile solutions for parenteral use.

The process of the invention for the preparation of compounds of general formula (I) involves the reaction of a histidylprolinamide of the desired optical configuration with the acid of the previously defined formula (II) in the presence of a solvent and a carbodidide, preferably dicyclohexylcarbodiimide, or with an acid chloride of formula (II).

The compounds obtained by the process of the invention are relatively stable and can therefore be purified, e.g., by precipitation, recrystallization, but also by column chromatography, counter current distribution, etc.

The following examples further illustrate the invention. The temperature data is not all corrected. Be careful not to make any examples of optimal pricing.

represents a group which may be 'hydrogenolytically cleaved', preferably a carbobenzoxy or substituted carbobenzoxy group; iii a functional acid derivative of formula II a, such as for example an acid halide, anhydride, or mixed anhydride, or an activated ester.

In the reaction of histidyl-prolinamide with an acid of formula II or Ha, or their derivatives, the 1- (3) position of the imidazole group in the hdstidyl moiety can be protected from the action of the acylating agent. Suitable protecting groups are known from peptide chemistry. These include, first of all, carbobenzoxir groups or substituted carbobenzoxy groups, trityl residues, o-nitrophenoxyacetyl residues, tert-butyloxycarbonyl groups, and other known residues which, since the reaction has occurred, , can be separated by hydrolysis by hydrogenolysis. Instead of histidyl-prolinamide with a possibly protected imidazole group, a silylated histidylprolmamide derivative, obtained by reacting histidyl-prolinamide with some trialkylsilanol or dialkylsilanediol (with 1 to 3 carbon atoms) in each of the alkyl groups, may be taken according to the invention. , which are derived in a known manner from a silylating agent; such as hexamethyldisilazane, trimethylchlorosilane, trimethylsilylacetamide, dimethyldichlorosilane and. t. d. After the complete reaction, which takes place in the presence of a solvent inactive to protons, the silyl groups can be cleaved by hydrolysis or alcoholism under mild conditions.

For the preparation of the compound of formula I _(it may be proceeded by the compound of formula III. '- S z ^R 3 N = |? F \ r ₂ NH ¹ r, ^{1 1 1} z ni ruj-, / ^c \ ^c \ | ²

0 ^Z CO - NH - CH - COOH ΠΙ.

X where R 1 to R ₃ and Z have the same meaning as above and X represents the hydrogen atom atom of the above defined group Y, in which the positions of the 1- (3) imidazole groups can be protected in the previously explained reversible manner, reacquires with prolinamide , in the presence of a water-binding agent, preferably one carbodiimide, preferably dicyclohexylcarbodiimide; iii. a functional derivative of one of the compounds of formula III, such as an acid halide, a tertiary anhydride, or an activated ester, is also reacted with pPOlinaittidohi, with the eventual separation of the protecting group from imidazolyl oetate.

The compounds of formula I may also be prepared by the compounds of formula IV and

i

i

X

R, CHj

CO - NH - CH - ca «H. Sesa.

IV wherein R 1 to R ₃ as well as X and Z have the same meaning as above and W denotes a hydroxyl or aryloxy group, a ρ-nitrophenoxy-, pyridyloxy-, a fertmercapto-, p-nitrophenylmercapto- or a cyanomethyloxy residue, or a residue N -hydroxy-succirtimide; This results in ammonia or some substance releasing ammonia under the reaction conditions, such as ammonium carbonate, as a fta example, and in this process it is possible to reversibly protect the position of the 1- (3) iftiidazalll-residue prior to the reaction in the described manner.

Orotyl-histidyl-prolinamide (in which one or both amino acids can be optically active or rooemic, and usually exist in the L-form) can also be particularly well obtained by reacting the desired optic configuration with the acid of the desired formula - 39 330

wherein Hal denotes a chlorine or bromine atom; in the presence of a water-binding agent, especially a carbodiimide (preferably di-cyclohexyl-carbodiimide), or is reacted with a functional acid derivative of formula V, such as an acid halide, anhydride, or mixed anhydride, azide, or activated ester, and then, in the further step of the process, the halogen atom is removed hydroganolitically from the intermediate obtained. ,

In the reaction of histidyl-prolinamide with an acid of formula V or a derivative thereof, the position of the 1- (3) imidazole-group u can be protected. histidyl residue, from the action of the acylating agent. Suitable protecting groups are those already mentioned above and the peptide chemistry used. Particular consideration is given to those (such as carbobenzoxy groups or substituted carbobenzoxy groups, or o-nitrophenoxyethyl ethyl moieties) which can be removed by hydrogenolytic.

In such cases, the separation of the protecting group from the first-step acylation product usually occurs at the same time as the hydrogenolysis of the halogen atom. Of course, the imidazole group in histidyl may also be bound to the residue by a known residue, separated by hydrolysis after reaction (for example, trityl derivative, tert. Butyloxycarboxyl group, etc.) and then, in the usual way, cleaved off and protecting groups, after the coupling reaction was completed, but only after the halogen atom hydrogenolysis was followed.

Hydrogenolysis of the halogen atom is carried out with the help of catalytically activated hydrogen, using it as a catalyst. it usually uses a precious metal catalyst for hydration (such as platinum and / or palladium on coal, barium sulfate, aluminum oxide, calcium or barium carbonate, and other known precious metal catalysts). Hydrogenolysis can be performed under normal or elevated pressure; usually at room temperature. Usually the solvent is water, iii mixtures of water and low alcohols (methanol, ethanol) or water and tetrahydrofuran, dioxane and the like. It may also work, for example, in glacial acetic acid as a solvent. Hydrogenolysis, which occurs during hydrogenolysis, is usually used to bind halogen hydrochloric acid in the presence of a hydrogen halide-binding substance. When using the aforementioned solvents or other non-acidically reactive solvents, halogenating agents, for example magnesium oxide, barium oxide, alkali metal hydroxides, ammonia or ammonium hydroxide, triethylamine, or salts of said substances with acids that are substantially lower than those of hydrochloric acid; 'such as carbonates or acetates of alkali metals. Also suitable for hydrogenolysis are, for example, alkali-, alkaline earth-, ammonium-, or amdnacetate solutions in acetic acid.

As mixed acid anhydrides, formulas II, II a, III, and V respectively, particularly those anhydrides derived from a carbonic acid monoester with aliphatic alcohols containing 1 to 4 carbon atoms, or for example derived from trimethylacetic acid acids (pivalic acids). Suitable activated esters of said acids are derived, for example, from p-nitrophenol, tri- or pentachlorophenol, pentafluorophenol, N-hydroxy-succinimide, 2- or 4-hydroxypyridine, thiophenol, p-nitrothiophenol, nitrile glycolic acid, 1-hydroxy- benzotriazole. and other hydroxy- or mercapto-type compounds of the usual peptide chemistry for the construction of activated esters.

The compounds obtained by the process of the invention are relatively stable, and therefore can be purified, e.g. precipitation, crystallization as well as column chromatography, counter current distribution, etc.

The following examples illustrate the invention. All temperature data is uncorrected. In performing these examples, no attention was paid to achieving optimal yields.

Example 1

15.6 g of orotic acid, 14.4 g of 1-hydroxy-benzotriazole and L-histidyl-L-prolinamide-dihydrobromide, obtained from 38.5 g of N-benzyloxycarbonyl-L-histidyl-L-propylamide (K. Inouye et al., Buli Chem. Soc. Soc. Japan, 44, 1689-91 (1971)) was added to 200 ml of dimethylformamide by the cleavage of the benzyloxycarbonyl group using a 40% solution of hydrobromic acid in glacial acetic acid. Cool to - 5 ° C and add, stirring,

27.8 ml of triethylamine, and immediately thereafter, at the same temperature, a solution of 20.6 g of Ν'-dicyclohexyl-carbodiimide in 50 ml of dimethylformamide. It is stirred for 24 hours and allowed to rise to room temperature. After that time, the precipitate formed was filtered off and the filtrate was evaporated under reduced pressure. The residue was added to 200 ml of water and stored at 0 ° C for 12 hours. After filtration, the filtrate was stirred at room temperature for 10 minutes with 170 g of the cation exchanger in free acid form, such as with Dow Chemical Cg. "Dowex-50 WX 4" (200 to 400 meshes), and then filtered. The cation exchanger, filled with the product, is well flushed with water, methanol and again with water. In the end, it mixes with oa. 200 ml of 1 n ammonia, filters, and resin - 39 330 -

-oxo-thiomorpholine-3-carbonyl-L-histidyl-L-prolinamide hydrate. Melting point 155-157 ° C; Ια / τΡ— = —48.5 ° (c = 0.52, methanol) was washed repeatedly with 1 n ammonia. The filtrate was evaporated under reduced pressure at temperatures up to 35 ° C to a volume of ca. 100 ml. The concentrated solution thus obtained is treated with activated charcoal and then frozen. Double boiling with 250 ml of absolute ethanol results in more extensive purification. For final purification, column chromatography may be used. To this end, they dissolve, for example,

2-3 g of the material in 5 ml of water, 10 ml of methanol is added to the entire column, and then all is sent over a 200 g base alumina column. Elution with methanol / water (2: 1) followed by ultraviolet absorption at 254 mm (= 2.54 cm ^-5 ). Impurities are eluted first, along with something major; followed by elution of the pure product. The combined fractions containing the main product were evaporated under reduced pressure. The residue was dissolved in water and frozen. 11.25 g = 28% of theoretical orotyl-L-histidyl-L-prolinamide dihydrate is thus obtained. Melting point 250 ° C; / a / D ²² = —45.8 ° (c = 1, methanol).

(407,4)

calculated C 47.20 H 5.19 N 24,12 found 47.95 5,27 24,10

The same product can also be obtained by reacting L-histidyl-L-prolinamide with ortho-acid chloride (J. med. Chem. 6, 334-335 (1963)), whereby usually ortho-acid chloride is released from ortho-acid, which is not translated into chloride by dissolving in absolute tetrahydrofuran.

Example 2

9.7 g of L-5-oxo-thiomorpholine-3-carboxylic acid (Tetrahedron, 28 4503 - 13 (1972)) and L-histidyl-L-prolinamide-dihydrobromide, obtained from 23.2 g of N-benzyl-oxycarbonyl- L-histidyl-L-prolineimide as in Example 1 was added to 150 ml of dimethylformamide. Cool to -10 ° C, stirred

16.7 ml of triethylamine, and immediately thereafter, at the same temperature, a solution of 12.3 g of Ν, Ν'-dicyclohexyl carbodiimide in 50 ml of dimethylformamide. It is stirred for 24 hours, allowing the temperature to rise to room temperature. After that, the precipitate was precipitated, the filtrate was evaporated under reduced pressure, and the residue thus obtained was added to 200 ml of water. After 2-časovnog holding at room temperature, the Procédé a filtrate treated katjonskim exchanger and at the place of 1 N ammonia solution, such as a described in Example 1. The j unique yen ammonium alkaline filtrate was evaporated under reduced pressure at temperature below 35 ^OC The rest is subjected to a counter current distribution in the n-butanol / water system. The fractions containing the main product are collected and evaporated under reduced pressure. The residue is dissolved in water, treated with charcoal and freezed. 6.9 g = 30% of theoretical L-5 Example 3 is obtained

5.1 g of imidazole-2-one-4-carboxylic acid, 5.8 g of 1-hydroxy-benzotriazole and L-histidyl-L-prolinamide, obtained from 15.4 g of N-benzyl-oxycarbonyl-L-histidyl-L -prolineamide of Example 1 was added to 100 ml of dimethylformamide. Cool to -5 ° C, add one for precious 11.1 ml of triethylamine and a solution of 8.3 g of N, N'-dicyclohexyl-carbodiimide in 30 ml of dimethylformamide. Leave for 30 minutes in an ice bath and then stir for 12 hours at room temperature. After that time, the precipitate formed is filtered off and the filtrate is evaporated under reduced pressure at ca. 35 ° C. To the residue was added 100 ml of water and allowed to stand at 0 ° C for 12 hours. After filtration, the filtrate was treated, as described in Example 1, with a cation exchanger and finally with 1 n ammonia; evaporates under reduced pressure and then freezes. The material thus obtained reverses the distribution of the opposite current in the n-butanol / water system. The combined fractions containing the main product were evaporated and the residue was recrystallized twice from 50 ml of methanol and 500 ml of chloroform each. Obtained after drying over phosphoropentoxide 4.5 g - 27% of theoretical imidazole-2-one-4-carbonyl-L-histidyl-L-prolinamide trihydrate. The substance of the powder is to sinter at 189 ° C, and it is further melted by heating at 212 ° C; / α / ϋ ²³ = —45.3 ° (c = 0.64, methanol).

CjsHnNA. 3H ₂ O (415,4) C H N calculated: 43.35 6.07 23.60 found: 43.66 5.35 23,59 Example 4

a) 12.0 g of L-cystne are dissolved in 300 ml of liquid ammonia. Add, with stirring, as much sodium (ca. 5.0 g) in small portions, while the dark blue color stays stable for 20-30 seconds, showing a slight excess of sodium. Addition of several ammonium chloride crystals discolored. Then, with stirring, 10.8 g of 2-chloropropionic acid amide is added. It was stirred for 10 minutes and then the ammonia was evaporated. The residue was dissolved in about 150 ml of water, and the pH of the solution was adjusted to 7 with concentrated hydrochloric acid. After filtration, the filtrate was evaporated under reduced pressure; the residue is dried over phosphorpentoxide, and then suspended in 200 ml of glacial acetic acid. The mixture is heated to boiling for 10 minutes. Glacial acetic acid is then evaporated under reduced pressure. The residue is extracted twice with 200 ml of hot methanol each. The single extracts were evaporated, ί osta6

330 i

it is dissolved in 50 to 100 ml of water. The pH of the solution is adjusted to approx. 2. The crystallization, which occurs spontaneously, is completed at 0 ° C. The crystals were purified and crystallized twice from 50 ml of water each. After drying over phosphoropentoxide, 6.6 g = 38% of the theoretical yield of thiomorpholine-5-one-6 (D, L) -methyl-3 (L) -carbonic acid are obtained. The product is pure enough for further reaction. For analysis, it was recrystallized twice more from 80 ml ethanol each and twice from 90 ml ethanol / water (1: 1) each to give the desired acid as white needles, melting point 186-188 ° C; Ζο, / d ²³ = + 37.7 ° (c = 1, methanol).

C ₆ H, NO ₃ S (175,2) calculated: C 41.35 H 5.17 N 7.98 S 18.30 found: 41.03 5.09 8.06 18.01

b) 17.5 g of thiomorpholin-5-one-6 (D, L) -methyl-3 (L) carboxylic acid, 14.4 g of 1-hydroxy-benzotriazole, and L-histidyl-L-prelinamide dihydrobromide, obtained from 38.5 g of N-benzyloxycarbonyl-L-histidyl-L-prolinamide according to Example 1 were dissolved in 200 ml of dimethylformamide. Cool to -5 ° C and add, one by one, with stirring, 27.8 ml of triethylamine and a solution of 20.6 g of N, N'-dicyclohexyl-carbodiimide in 30 ml of dimethylformamide, leave for 1 hour in an ice bath (- 5 ° to 0'C) and then allowed to rise to room temperature, with a stirring time of 12 hours. After that time, the precipitate formed formed. The filtrate was evaporated under reduced pressure at 35 ° C. The residue was mixed with 150 ml of water and stored at 0 ° C for 12 hours; the filtrate was then filtered and, as described in Example 1, treated with a cation exchange resin followed by 1 n ammonia. The filtrate evaporates under reduced pressure, and finally freezes. It was then recrystallized twice from 75 ml of water each, once from 50 ml of ethanol / water (1: 1) and then three times from 50 ml of water saturated n-butanol each. Drying over phosphorpentoxide afforded thiomorpholine-5-one-6 (D, L) rniethyl-3 (L) -carbonyl-L-histidyl-L-prolinamide-trihydrate. Recrystallization of the remainder of the collected and evaporated mother liquors, which yields further amounts of this compound, The total yield then reaches 14.5g = 31.4% of theory. Melting point 140-142 ° C; ΙαΙτΡ = —33.5 ° (c = l, methanol).

CpHaNAS. 3H10 (462.5) in Example 4, in 500 ml of liquid ammonia. The reaction product, dried in vacuo above phosphorpentoxide, was heated with 140 ml glacial acetic acid until boiling for 15 minutes. The glacial acetic acid was then evaporated under reduced pressure and the residue dissolved in 60 ml of water. The pH of the solution was adjusted to · ca. 2 with 6 n hydrochloric acid. The crystallization occurring at the bottom is completed at '0 ° C. The crystalline substance is filtered off and recrystallized from 60 ml of water. Drying over phosphorus pentoxide in vacuo afforded 7.0 g (74%) of D-thio-morpholine-5-one-2,2-dimethyl-3-carboxylic acid. Melting point 189-192 ° C; / α / ϋ ²³ = —14.2 ° (c - 1, methanol).

C ₇ H _n NO ₃ S (189,239) calculated: C 44.35 H 5.82 N 7.41 S 16.93 found: 44.82 5.41 7.92 17.14

b) 13.2 g of D-thiomorpholine-5-one-2,2-dimethyl-3-carboxylic acid, 10.1 g of 1-hydroxy-benzotriazole, from 38.5 g of benal-oxycarbonyl-L-histidyl-L- of prolinamide, obtained according to Example 1, L-histidyl-L-prolinamide-dihydrobremide, 27.8 ml of triethylamine, as well as 14.4 g of N, N'-dicyclohexyl-carbodhmidide were reacted analogously to Example 4b, and treated analogously until, finally, a purification operation using a cation exchanger and freezing. In this way the material obtained is subjected to a counter current distribution in the n-butanol-water system. The fractions containing the main product are evaporated under reduced pressure. Of which, 2 g were dissolved in methanol-water (1: 1) and chromatographed over a column prepared from 350 g of basic alumina; whereby elution is monitored by ultraviolet absorption at 254 nm. The fractions containing the pure product were evaporated under reduced pressure. The residue was dissolved in water and crystallized.

In this way 7.6 g = 24.8% of the theoretical yield, calculated on the applied acid, of D-thiomorpholine-5-one-2,2-dimethyl-3-carbonyl-L-histidyl-L-prolinamide hydrate is obtained.

Melting point: 153-155 ° C; / <z / d ²⁴ = -83 ° (c = '1, methanol).

C ₁₈ H2aN ₆ O ₄ S .. H ₂ O (440,536) calculated:

found:

C H

44.15 655

44.69 6.47

N S

18.17 6.93

1852 654

calculated: C 49.07 H 6.41 N 19.08 S 7.28 found: 48,80 6.43 19.08 7.24

Example 5

a) 75 g of D - (-) - penicillamine, 2.9 g of sodium and

5.2 g of chloracetamide were reacted as described

Example 6

17.0 g of 5-methyl-orotic acid and 14.4 g of 1-hydroxybenzotriazole are added to 100 ml of dimethyl-39 330-formamide, and stirred at room temperature with stirring, with 27.8 ml of triethylamine After 10 minutes. it is cooled in an ice bath, and after a further 10 minutes, stirring is added

20.6 g of N, N'-dicyclohexyl-carbodiimide in 30 ml of dimethylformamide followed by L-histidyl-L-prolinamide-dihydrobromide - obtained from 38.5 g of phenyl-oxycarbonyl-L-histidyl-L-prolinamide, such as described in Example 1- and 100 ml of dimethylformamide. After that, it was stirred for 0.5 hours in an ice bath and 12 hours at room temperature. Separation is performed as described in Example 1, including pre-treatment on a cation exchanger. The crystallized material thus obtained is dissolved in 110 ml of ethanol. After 24 hours at 0 ° C, the product is dried and the product is dried directly in vacuo over phosphorous. 2 g of this substance were dissolved in methanol-water (1: 1) and chromatographed over a column prepared from 350 g of basic alumina, whereby the elution of the individual fractions was monitored by ultraviolet absorption at 254 nm. The fractions containing the pure product were evaporated under reduced pressure. The residue was dissolved in water and crystallized. This gives 14.4 g = 30.4% of the theoretical yield of 5-methyl-orotyl-L-histidyl-L-prolinamide-trihydrate.

Melting point: 222 - 227 ° C (decomposition); / oc / d ²⁴ = -46.8 ° (c-1, methanol).

C ₁₇ H ₂₁ N ₇ O ₅ . 3H ₂ O (457,459) C H N calculated: 44.63 5.95 21,43 found: 44.67 5.58 21,79 Distant quantities desired products can be profit from paired of residues mother liquors to and from

prefraction using the chromatography described above.

Example 7

Proceed as in Example 6 except that 18.4 g of 5-ethyl-orotic acid is used instead of 5-methylonotinic acid. The reaction products were separated as described in Example 6, including pre-treatment with a cation exchanger.

The crystallized product thus obtained undergoes a counter current distribution in the n-butanol-water system. The fractions containing the desired product were evaporated under reduced pressure, dissolved in water, and crystallized. It is now crystallized twice from 20 ml of water each. After drying in vacuo over phosphorpentoxide, 12.3 g = 28.3% of the theoretical yield of 3-ethyl-orotyl-L-histddyl-L-prolinamide-hydrate is obtained. Melting point 186 ° C; / α / ϋ ²⁴ = - —46.0 (c = 0.5, methanol).

CisHaNA · H ₂ O (435,427). C H N calculated: 49,64% Wins 5,79% Draw 22,52% found: 49.50% 5.87% 22,59% Stem the puddles after crystallization are scoured -

Thus, a further 4.1 g (9.4%) were obtained, all in all, 16.4, g = 37.7% of the theoretical yield of the desired product.

Example 8

Proceed as in Example 6, but 19.8 g of 5-n-propyl-orotic acid is used instead of 5-Methyl-Orotic acid. After separation of the reaction products and before purification using a Caiton exchanger according to the procedure described in Example 12, the crystallized material obtained is dissolved in 90 ml of ethanol. After 12 h storage at 0 ° C, the gelatinous mass is mixed with 100 ice ethanol and the procedures. Recrystallization from methanol / ethanol (1: 5) and water methanol (1: 1) afforded pure 5-n-propyl-orotyl-L-hisitidyl-L-prolinamide hydrates. The mother liquor was evaporated and the residue was recrystallized from methanol-water (1: 1), methanol ethanol (1: 5) and then again from methanol water (1: 1). In this way, further quantities of the desired product are obtained

Total yield 17.1 g = 38.0% of theory. Melting point 187 ° C; / oc / d ²⁴ = -48.6 ° (c = 0.5

methanol). σ ,, Η ^ ΝΑ.ΗζΟ (449,493) C H N Calculated: 50.77% 6.06% 2132% Found: 5036% 6,12% 22,19% According to the above, especially according to from

The following compounds of general formula (I) may also be obtained by examples of apparent modes (provided that no configuration information is provided, the starting components in all these compounds, i.e., prolinamide, hdstidine, and xfselin of formula (II) may exist as racemates, in L-form, or in D-form, however, in all cases, L-config is preferred).

Orotyl-D, L-histidyl-D, L-prolinamide

Orotyl-L-histidyl-D-prolinamide

Orotyl-L-histidyl-D, L-prolinamide (5-Iso-propyl-orotyl) -histidyl-prolinamide

5-Oxo-2,2,6-trimethyl-thiomorpholine-3-carbonyl-histidyl-prolinamide s

- 39 38fl

5-Oxo-2,3,6-trimethyl-thiomorpholin-3-carbQnit-histidyl-propylnamide

5-Oxo-6,6Khraethyl-thiomorpholine-3-carbonyl-histidyl-prolinamide.

R4,

I _G — St—

I

Rs

The Applicant hereby declares that, as far as he is aware, the best way to economically use the invention is to use the method of Example 1 contained in the text of the description of this application.

Claims (4)

PATENT REQUIREMENTS 1. A process for preparing a dipeptide derivative of the general formula: £ 1. I: H. ^Xr 2 ' ^X CQ in which Ri and R2 are the same. iii, understood ;, and conceived: hydrogen atoms, alkyl. residues with 1–3 Ca borons, III or conjunctivities, form an additional bond between the G-atoms which carries these residues, R _3l jp _: atom. hydrogen or an alkyl residue having 1 to 3 C atoms, Z. is a group formula: OH I '_N = C— or. -NH-CO-, iii ui kojoj su R 4 and R _5th the same iii different and denoting hydrogen iii. alkyl groups with 11—3 Chatema, as ifanrnaeeudskl · acceptable · addition salts ovdh. compounds, indicated; in that histidylpralinamide reacts with. Carboxylic Acid Formula: H 'in which R ·, up to H ₃ and · Z ^r have the upper meanings, at a temperature of approx. -1 £). ° C to. at room temperature, in the presence of a solvent, preferably dimethiformamide and in the presence of carbodiimide, or iii with a halogenated home of an acid of formula (H). 2. Method for obtaining orotilhistidilpropilamina axle zahtevu 1, is indicated · time, a hundred histidilprolinamid reacted with an acid orotnom ^{j 1} in the presence of a carbodiimide. The process according to claims 1 and 2, characterized in that as. carbidiimide uses dicyclohexylcarbodiimide. 4. The method of claim 1, wherein; which is used as an acid halide of formula (II). Publisher: Federal Patent Office, Belgrade, Uzun Mirkova 1 Printed by: IPP »BANAT« OOUR »Graphics« Kikinda, B. Kidrica 54