NO752947L - - Google Patents

Description

AMIDE

The present invention relates to a new tetrapeptidamide of the formula

in which all the amino acids with a center of asymmetry

exhibits the L configuration,

and its acid addition salts, as well as methods for producing these compounds.

The abbreviations used in the context of the present invention for the individual amino acids and their protecting groups are the abbreviations that are now common in peptide chemistry and are generally known to those skilled in the art (literature: Schroder E. und LUbke, K., The Peptides, Academic press, New York & London, Vol. I (1965) and Vol. II (1966) and IUPAC-IUB Rules),

they therefore need no further definition here, with the exception of "pGlu" which should mean pyroglutamic acid.

Examples of acid addition salts of the compound of formula I

are salts with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, perchloric acid and the like or with organic acids such as acetic, oxalic, maleic, malic, tartaric or citric acid and the like. Particularly preferred are the pharmaceutically usable non-toxic acid addition salts.

The term "lower alkyl" in the context of the present invention means straight-chain or branched 'hydrocarbon residues with 1-6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl,

pentyl, hexyl, etc.. The term "aryl" means especially the phenyl radical; which may be substituted or unsubstituted. Substituents for the phenyl radical are especially lower alkoxy, nitro, halogen such as fluorine, chlorine, bromine and iodine. Examples of substituted phenyl residues are p-nitrophenyl, tri- or pentachlorophenyl and the like. "Lower alkoxy means a lower alkyl-oxy group, in which the lower alkyl residue has the above meaning, such as methoxy, ethoxy, propoxy, butoxy etc. ♦

The new tetrapeptidamide of formula I and its acid addition salts can be prepared in a manner known per se, by

a) converts the dipeptide of the formula

with a dipeptide of the general formula

in which produces hydroxy or a residue that activates the carboxyl group,

or that one

b) converts a tripeptide of the general formula

wherein R1 has the above meaning,

with the amide of the formula

or that one

c) converts the tripeptide of formula

. with

in which the above has the meaning,

or that one

d) cleaves off the protecting group(s) of a tetrapeptide of the general formula

wherein R^ represents hydrogen or an amino protecting group,

R^ means hydrogen or a group protecting the imidazole function and

R^ hydrogen or an amide protecting group, whereby at least one of the residues R 2 , R 3 and R^ is different from hydrogen,

or that one

e) amidates a tetrapeptide of the general formula

in which X produces hydroxy, a residue that activates

the carboxyl group or the residue -OR=,.* , where R means lower alkyl, aryl, aryl-lower alkyl or the residue of a polymeric support material,

or that one

f) in a tetrapeptide of the general formula

in which R produces the optionally protected glutamine or glutamic acid residue,

Y a residue which activates the carboxyl group, the residue -NHR^, in which R^ has the meaning indicated above, or the residue -OR^, in which R^ has the meaning indicated above,

during simultaneous or previous spin-off of any j

occurring protecting groups close the pyroglutamine ring, and that, if desired, one transfers an obtained compound of formula I into an acid addition salt, whereby all the amino acids with an asymmetric center in formulas II to X exhibit the L configuration.

Examples of groups that activate the carboxyl group are

esters such as cyanomethyl-, p-cyanophenyl-, p-nitrophenyl-, 2,4,5-trichlorophenyl-, pentachlorophenyl-, thiophenyl-, p-nitrothiophenyl-, 1- benztriazolyl-, phthalimidyl-, 1-succinimidyl-, 1- piperidyl, 8-quinoly<*>1-, 5-chloro-8-quinolyl-, 2-pyridyl-, 2-thiopyridyl esters, azides and halides.

It can be used in all contexts with peptide syntheses

known protective groups.

Examples of amino protecting groups are those of the acyl type

( such as formyl, benzoyl, phthalyl, trifluoroacetyl, p-tosyl, aryl-

and alkylphosphoryl, phenyl- and benzylsulfonyl, tritylsulfenyl, o-initrophenylsulfenyl,/"-chlorobutyryl or o-nitrophenoxyacetyl), of the alkyl type (such as trityl, benzyl, alkylidene) or of the urethane type (such as carbobenzoxy, p-bromo- , p-chloro- or p-methoxycarbo-benzoxy, tolyloxy-, allyloxy-, cyclopentyloxy-, cyclohexyloxy-, t-butyloxy- or 1,1-dimethylpropyloxy-, 2-(p-biphenylyl)-2-propyloxy- carbonyl or benzylthiocarbonyl), etc.

Examples of amide protecting groups are xanthenyl, 2,4-dimethoxybenzyl, 2,4,6-trimethoxybenzyl and 4,4<1->dimethoxybenzhydryl and the like.

Examples of carboxyl protecting groups are O- and S-esters

(as methyl-, ethyl-, t-butyl-, benzyl-, cyanomethyl-, phthalimido-methyl-, 4-picolyl-, 2-p-tosylethyl-, phenyl-, p-nitrophenyl-, thiophenyl- or nitrobenzyl ester), amides or hydroazides (such as trityl, phenyl, carbobenzoxy or t-butoxycarbonyl hydrazides). Furthermore, the carboxyl group can be protected by salt formation. Also the residue of a polymer support can be considered as a carboxyl protecting group.

As special protecting groups for the histidine residue can

examples are mentioned: benzyl, p-nitrobenzyl, tert. butoxycarbonyl^

tosyl, piperidinocarbonyl and the like.

The reaction as well of the dipeptide of formula II with a dipeptide

of formula III, which also. of a tripeptide of formula IV with the amide of formula V as well as that of the tripeptide of formula VI with a compound of formula VII can be carried out in a manner known per se. Appropriately, this turnover takes place in a

inert organic solvent, preferably in a polar organic solvent, such as about dimethylformamide, dimethylsulfoxide, acetonitrile, halogenated hydrocarbons,

such as dichloromethane, chloroform and the like. Reactions also conveniently take place at a temperature below room temperature. If the residue in the dipeptide of formula III or in et

tripeptide of formula IV produces the hydroxy group,

the reaction conveniently takes place in the presence of a condensing agent, such as dicyclohexylcarbodiimide, carbonyldiimidazole and the like or also according to the mixed anhydride method.

The removal of the protective group(s) from a peptide of formula VITI or X can likewise take place in a generally known manner and under the reaction conditions applicable to the individual groups. Thus, e.g. the cleavage of a benzyloxycarbonyl group takes place by hydrogenation in the presence of a catalyst, for example a palladium/carbon catalyst. The tertiary butyloxycarbonyl group can e.g. removed by treatment with trifluoroacetic acid or with HCl in glacial acetic acid.

The amidation of a tetrapeptide of the general formula IX can likewise take place in a manner known per se: preferably by reaction with ammonia at room temperature and in a suitable organic solvent.

The termination of the pyroglutamine ring in a tetrapeptide of the formula

X can likewise be carried out in a manner known per se. Thus, for example, the ring closure of a compound of formula X, in which R fi produces a glutamic acid residue protected with an ester group and Y the residue -OR^, with simultaneous removal of the protective groups, by treatment with ammonia.

in

They used the compounds of the formulas as starting material

II, VI, VIII, IX and X are new compounds and likewise

subject of the present invention as such.

The production of the new output connections can be carried out

in a manner known per se using the usual, especially the above-mentioned protecting groups.

The compound of formula I according to the invention and its pharmaceutically usable acid addition salts have antidepressant action and can be used for the treatment of depressive syndromes.

The effect of antidepressants can be explained by the fact that these

Pharmaka normalizes the activity of the brain's noradrenergic neurons, which are altered in depressive disorders.

The change in the activity of noradrenergic neurons can be seen by measuring the norepinephrine (NA) release, e.g. by determining the concentration of endogenous NA in the brain after blocking NA synthesis by dopamine-(3-hydroxylase inhibitor bis-(4-methyl-1-homo-piperazinyl-thiocarbonyl-disulfide) (FLA 63). Has a precursor substance NA- liberating effect, this is shown in a strengthening of FLA 63-induced NA lowering.

To determine the antidepressant effect of the compound according to the invention, the lowering of NA in the whole brain of male mice is determined 90 minutes after i.p. injection of FLA 63 (5 mg/kg). The value found after FLA 63 alone is indicated as relative loo% in the table. Simultaneous injection of L-pyroglutamyl-L-histidyl-L-prolyl-p-alanine amide (test substance) forte

to a significant acceleration of the FLA-63 induced NA lowering.

Accordingly, the compound of the invention increases the release of NA in the mouse brain. This result and the observed increase in the locomotor activity of these mice is proof of the antidepressant effect of this preparation. L-pyroglutamyl-L-histidyl-L-prolyl-(3-alanine amide) has an LD lo of over 8 g/kg p.o. and an LD 5o of over 4 g/kg i.v. in the mouse.

The dosage must be regulated according to individual needs and can vary from loo \ iq/ to 1 mg i.v. or from lo to loo mg p.o. per single dose, administered one to several times a day.

The process product of formula I as well as its pharmaceutically usable acid addition salts can all find use as pharmaceuticals in the form of pharmaceutical preparations, which contain these products in admixture with a pharmaceutical, organic or inorganic inert carrier material suitable for enteral or parenteral application, such as e.g. water, gelatin, gum arabic, milk sugar, starch, magnesium stearate, talc, vegetable oils, polyalkylene glycol, vaseline, etc. The pharmaceutical preparations can be in solid form, e.g. as tablets, dragees, suppositories, capsules, or in liquid form,

e.g. as solutions, suspensions, syrups or emulsions. If necessary, they are sterilized and resp. or contains auxiliary substances such as preservatives, stabilisers, cross-linking

or emulsifiers, salts for changing the osmotic pressure or puffs. Preferred forms of administration are solutions (ampoules), tablets and intranasal spray solutions.

EXAMPLE 1

a) p-alanine methyl ester hydrochloride.

22 g (3-alanine was heated in 4N HCl in methanol over the course of 1 hour

at reflux and then evaporated in vacuo to dryness.

This treatment is repeated 2 times and the remaining

the oil was crystallized from methanol/ether. Yield: 27.5 g,

Bp 92°C.

b) t-butyloxycarbonyl-L-prolyl-p-alanine methyl ester.

17.2 g of t-butyloxycarbonyl-L-proline was dissolved in 160 ml of tetrahydrofuran, cooled to -2o°C and 11.1 ml of triethylamine and 7.7 ml of ethyl chloroformate were added. The suspension was stirred for 5 min.

to at -2o°C and added a suspension precooled to -2o°C, prepared from 13.4 g of [B-alanine methyl ester hydrochloride and 13.4 ml of triethylamine in 16o ml of a mixture of dimethylformamide and tetrahydrofuran (1:1) . The reaction mixture was stirred for 1 hour below -10°C and another hour at room temperature, filtered and the filtrate evaporated in vacuo. The oily residue was dissolved in 200 ml of ethyl acetate and washed three times each with 5% KHSO 4 solution and water and dried over Na 2 SO 4 . The organic phase was evaporated in vacuo and the oily residue crystallized from ether/petroleum ether. Yield: 19.2 g, Fp 7o°C, /q7<25>= -39.2°

(c = 1,dimethylformamide). D

c) t-butyloxycarbonyl-L-prolyl-p-alanine amide.

A solution of 11 g of t-butyloxycarbonyl-L-prolyl-(3-alanine methyl ester in 80 ml of methanol was saturated with ammonia-

gas at 0° and stored for 2 days at room temperature. The solution was concentrated in vacuo and the residue was crystallized from methanol/water. Yield: 9.8 g, mp 175-177°C, /q/p<5>= -44.5° (c=1, dimethylformamide). d) L-pyroglutamyl-L-histidyl-L-prolyl-p-alanine amide acetate.

HCl gas was passed through a solution over the course of one hour

of 9 g of t-butyloxycarbonyl-L-prolyl-p-alanine amide in 50 ml of glacial acetic acid, the solution was concentrated in vacuo and the resulting,

The L-prolyl-[3-alanine amide hydrochloride was dried in vacuo overnight.

A suspension of 7.64 g of L-pyroglutamyl-L-histidine hydroazide in a mixture of 95 ml of dimethylsulfoxide and 125 ml of dimethylformamide,

was added at -2o°C with 104 ml of 1.64 N HCl in tetrahydrofuran.

To this solution was added 5 ml of isoamyl nitrite. The mixture was stirred for 30 min. at -2o°C, cooled to -3o°C neutralized at this temperature with 23.7 ml of triethylamine. 7.7 g of L-prolyl-p-alanine amide hydrochloride was dissolved in a mixture of 20 ml of dimethylsulfoxide* and 15 ml of dimethylformamide, cooled to -10°C and neutralized with 4.9 ml of triethylamine and added to the reaction mixture described above. The mixture thus obtained was stirred for 1 hour at -2o°C, stored at 4°C for 24 hours and filtered. The filtrate was evaporated in vacuo, the residue taken up in 35o ml of water

and extracted five times with each 7o ml of ethyl acetate. The aqueous phase was evaporated in vacuo and the residue purified by countercurrent distribution ± the system 1-butanol:glacial acetic acid:water (4:1:5). L-pyroglutamyl-L-histidyl-L-prolyl-(3-alanine amide acetate) was recovered by concentration of the corresponding fractions in vacuo and then lyophilization. /a/D 25 = -83.4 o( c = 1, in ln acetic acid).

After selection, the residue of the ethyl acetate extraction was dissolved

in 2o ml glacial acetic acid, added 3o ml acetic anhydride and kept overnight at room temperature. The reaction solution was then diluted with 500 ml of water and evaporated to dryness in vacuo.

The residue was taken up three more times with 50 ml each of water and again evaporated to dryness. The residue thus obtained was dissolved in 50 ml of water and chromatographed on Amberlite CG-50 (H+<-> form). L-pyroglutamyl-L-histidyl-L-histidyl-L-prolyl-(3-alanine amide) was eluted with 1N acetic acid. The corresponding fractions were combined and lyophilized.

Paper electrophoresis: buffer from 2 ml glacial acetic acid and 20 ml pyridine, made up to 1 liter with water (pH 6): Rf(histidine) = o.73- o.o5. Buffers from 37 ml of formic acid and 25 ml of glacial acetic acid, made up to 1 liter with water (pH =1.7): Rf(histidine)

= o.43 - o.o5.

EXAMPLE 2

1.1 g of benzyloxycarbonyl-(3-alanine amide) was hydrated in 25 ml

methanol while adding palladium/carbon. The catalyst was

filtered off and the filtrate evaporated in vacuo. The residue was dissolved in 10 ml of dimethylformamide, cooled to 0°C and 1.8 g of L-pyroglutamyl-L-histidyl-L-proline, 0.6 g of N-hydroxysuccinimide and 1.1 g of dicyclohexylcarbodiimide were added. The reaction mixture was stirred for 24 hours at room temperature, filtered and the filtrate evaporated in vacuo. The residue was chromatographed on silica gel in the system chloroform:methanol:water (6o:3o:5).

The thin-layer chromatographic uniform fractions were combined and evaporated in vacuo. The residue was dissolved in 0.1 N acetic acid and lyophilized. 1.8 L-pyroglutamyl-L-histidyl-L-- 25 o is obtained

prolyl p-alanine amide acetate. l. °J- q ~ -81/2 ( c = 1, in ln acetic acid).

EXAMPLE 3

a) Benzyloxycarbonyl-p-alanine amide

111.7 g of benzyloxycarbonyl-(3-alanine) were dissolved in 1 liter

dimethylformamide, cooled to -2o°C, added 7o ml of triethylamine and 67 ml of chloroformate isobutyl ester and stirred for 3 minutes at -2o°C. In this reaction mixture, dry ammonia-

gas introduced until the pH of the mixture rose to 11, whereby care was taken that the temperature did not exceed -lo°C. The reaction mixture was stirred for an additional 30 min. at room temperature, filtered and the filtrate evaporated in vacuo. The residue was recrystallized from water. Yield: 91 g, mp 162-165°C.

b) Benzyloxycarbonyl-L-prolyl-(3-alanine amide).

44.5 g of benzyloxycarbonyl-(3-alanine amide) was hydrated in 250 ml

methanol while adding palladium/carbon. The catalyst was filtered off and the filtrate evaporated in vacuo. The residue was suspended in 100 ml of dimethylformamide and 74.2 g of benzyloxycarbonyl-L-proline-p-nitrophenyl ester were added. The reaction mixture was stored for 24 hours at room temperature and evaporated in vacuo. The solid residue was recrystallized from ethyl acetate. Yield: 57.8 g, mp 151-152°C./α/25=-51.7° (c = 1, methanol).

c) Benzyloxycarbonyl-L-histidyl-L-prolyl-p-alaninamide II, 3 g of benzyloxycarbonyl-L-prolyl-(3-alaninamide) were dissolved in

2oo ml of methanol and hydrated with the addition of palladium/

carbon. The catalyst was filtered off and the filtrate evaporated in vacuo. The residue was dissolved in 100 ml of dimethylformamide. To a suspension of 10.7 g of benzyloxycarbonyl-L-histidine hydrazide in 150 ml of dimethylformamide was added at -2o°C 58.7 ml of 3 n HCl

in tetrahydrofuran. To this solution was added 6.7 ml of isoamyl nitrite. The mixture was stirred for 3 min. at -2o°C, cooled to -3o°C and neutralized with 24.6 ml of triethylamine at this temperature, and added to the -2o°C above-described solution of L-prolyl-P-alaniramide. The mixture thus obtained was stirred for 1 hour at -2o°C and stored for 24 hours at 4°C and filtered. The filtrate was evaporated in vacuo and the residue chromatographed on silica gel in the system chloroform/methanol (4:1). The thin-layer chromatographic uniform fractions were combined and evaporated in vacuo. The residue is crystallized from ethanol/ether. Yield: 12.9 g, Fp. 95-97°C, A725

Zq/^ = _40.8° (c = 1, methanol).

d) L-pyroglutamyl-L-histidyl-L-prolyl-p-alanine amide acetate.

1, and g benzyloxycarbonyl-L-histidyl-L-prolyl-p-alanine amide was

dissolved in 50 ml of methanol and hydrated with the addition of palladium/carbon. The catalyst was filtered off and the filtrate evaporated in vacuo. The residue was dissolved in 10 ml of dimethylformamide and 0.9 g of L-pyroglutamic acid pentachlorophenyl ester was added. The reaction mixture. was stored for 24 hours at room temperature and evaporated in vacuo. The residue was dissolved in water and chromatographed on a Dowex-2 soil (OH form). The thin-layer chromatographic uniform fractions were combined, acidified with acetic acid and lyophilized. Yield: o.8 g. /a/^<5>= -82.2° (c = 1, in ln acetic acid).

EKS EMPEL 4

■ a) Benzyloxycarbonyl-(3-alanine amide).

111.7 g of benzyloxycarbonyl-(3-alanine) was dissolved in 1 liter of dimethylformamide, cooled to -2o°C, 70 ml of triethylamine and 67 ml of chloroformate isobutyl ester were added and stirred for 3 minutes at -2o°C.

Into this reaction mixture, dry ammonia gas was introduced to pH until the mixture rose to 11, whereby care was taken that the temperature did not exceed -10°C. The reaction mixture was stirred for an additional 30 min. at room temperature, filtered and the filtrate was evaporated in vacuo. The residue was recrystallized from water. Yield: 91 g, Fp. 162-165°C.

b) Benzyloxycarbonyl-L-prolyl-(3-alanine amide).

44.5 g of benzyloxycarbonyl-p-alanine amide was hydrated in 25o ml

methanol while adding palladium/carbon. The catalyst was filtered off and the filtrate evaporated in vacuo. The residue was suspended in 100 ml of dimethylformamide and 14.2 g of benzyloxycarbonyl-L-proline-p-nitro-phenyl ester was added. The reaction mixture was stored for 24 hours at room temperature and evaporated in vacuo. The solid residue was recrystallized from ethyl acetate. Yield: 57.8 g, mp 151-152°C, Zq7^5 = -51.7° (c = 1, methanol).

c) Benzyloxycarbonyl-L-histidyl-L-prolyl-p-alanine amide.

11.3 g of benzyloxycarbonyl-L-prolyl-p-alanine amide was dissolved

in 200 ml of methanol and hydrated while adding palladium/carbon. The catalyst was filtered off and the filtrate evaporated in vacuo. The residue was dissolved in 100 ml of dimethylformamide. To a suspension of 10.7 g of benzyloxycarbonyl-L-histidine hydrazide in 150 ml of dimethylformamide was added at -20°C 58.7 ml of 3N HCl in tetrahydrofuran. To this solution was added 6.7 ml of isoamyl nitrite. The mixture was stirred for 30 min. at -2o°C, cooled to -3o°C and neutralized at this temperature with 24.6 ml of triethylamine and added with the solution of L-prolyl-(3-alanine amide precooled to -2o°C, as described above. The thus obtained the mixture was stirred for 1 hour at -2o° C., stored for 24 hours at 4° C. and filtered.The filtrate was evaporated in vacuo and the residue chromatographed on silica gel in the system chloroform:methanol (4:1).

The thin-layer chromatographic uniform fractions were

combined and evaporated in vacuo. The residue was crystallized from ethanol/ether.

Yield: 12.9 g, Fp. 95-97°C. / aj^ 5 = -40.8°, (c = 1, methanol) . d) Benzyloxycarbonyl-L-pyroglutamyl-L-histidyl-L-prolyl-(3-alanine amide hemiacetate).

1.0 g of benzyloxycarbonyl-L-histidyl-L-pyrrolyl-(3-alanine amide) was dissolved in 50 ml of methanol and hydrogenated with the addition of palladium/carbon. The catalyst was filtered off and the filtrate

evaporated in vacuum. The residue was dissolved in 10 ml of dimethylformamide, cooled to 0°C and 1 g of benzyloxycarbonyl-L-pyroglutamic acid p-nitrophenyl ester was added. The reaction solution was stored for 24 hours at room temperature and evaporated in vacuo. The rest stayed

dissolved in a mixture of chlorophorrum ethanol: glacial acetic acid (95:5:3)

and added a silica gel column. First, the soil was washed with this mixture, then the desired peptide was eluted with the system chloroform:methanol:water (6o:3o:5). The thin-layer chromatographically uniform fractions were combined and evaporated in vacuo.

The remainder was dissolved in 50 ml of water and lyophilized. Yield: o.9 g,

..25 o

LSy- Q = -11/° ( c = 1, ln acetic acid).

e) L-pyroglutamyl-L-histidyl-L-prolyl-(B-alanine amide acetate).

loo mg of benzyloxycarbonyl-L-pyroglutamyl-L-hustidyl-L-prolyl-(3-alaninamide hemiacetate) was dissolved in 20 ml o.5n acetic acid and hydrated with the addition of palladium/carbon. The catalyst was filtered off and the filtrate lyophilized. Yield: 8mg .Zq7D 25 = -82.5° ( c = 1, in ln acetic acid).

EXAMPLE 5

a) Benzyloxycarbonyl-L-prolyl-p-alanine methyl ester.

24.9 g of benzyloxycarbonyl-L-proline was dissolved in 200 ml of dimethylformamide, cooled to -20°C and 11.1 ml of N-methylmorpholine and 13.0 ml of chloroformate isobutyl ester were added. The suspension was stirred for a further 2 min. at -2o°C and added a suspension precooled to -2o°C, prepared f of 14.0 g (3-alanine methyl ester hydrochloride and 11.1 ml of N-methylmorpholine in 100 ml of dimethylformamide. The reaction mixture was stirred for 3o min. under -10°C and an additional 30 min at room temperature, filtered and the filtrate evaporated in vacuo. saturated NaCl solution and dried over Na^SO^ The organic phase was evaporated in vacuo and the oily residue crystallized from ethyl acetate/hexane Yield: 22.8 g, mp 55°C,

25 o

/q7D = -50.7 (c = 1, methanol).

j

i b) L-pyroglut amy 1-L-histidyl-L-prolyl-(3-alanine methyl ester acetate).

lo, and g benzyloxycarbonyl-L-prolyl-(3-alanine methyl ester) was

dissolved in 50 ml of methanol and hydrated with the addition of palladium/carbon. The catalyst was filtered off and the filtrate evaporated in vacuo. The remaining L-prolyl-(3-alanine methyl ester) was dissolved in 20 ml of dimethylformamide and reacted with L-pyroglutamyl-L-histidine azide (prepared from 8.4 g of L-pyroglutamyl-L-histidine hydrazide). The reaction mixture was stirred for 1 hour at -2o°C, stored, at 4°C and filtered. The filtrate was evaporated in vacuo, the residue taken up in 25o ml of water and extracted five times with each 100 ml of ethyl acetate. The aqueous phase was passed over a Dowex 5oW (H-shape) soil (3x35 cm), it was washed with 1 liter of water and the substance eluted with a buffer consisting of pyridine:glacial acetic acid:water (15o:2o:33o).The substance-containing fraction was diluted with water, allowed to to pH 5 with acetic acid, concentrated in vacuo, then again diluted with water and lyophilized. The lyophilisate was chromatographed on silica gel in the system chloroform:methanol:water (6o:3o:5). The thin-layer chromatographically uniform fractions were combined and evaporated in vacuo. The residue - 25 was dissolved in 2oo ml of water and lyophilized.Yield: 8.7 g, /q/

-82.8° ( c = 1, ln acetic acid). D

c) L-pyroglutamyl-L-histidyl-L-prolyl-p-alanine amide.

1.0 g of L-pyroglutamyl-L-histidyl-L-prolyl-(3-alanine methyl ester) acetate was dissolved in 100 ml of methanol. This solution was saturated at 0°C with ammonia, sealed gas-tight and stored for 3 days at room temperature. The solution was evaporated in vacuo, the residue dissolved in 5d ml of water and lyophilized. Yield: 9oo mg. /o/^<5>= -94.9° (c = 1, ln acetic acid).

IN

EXAMPLE 6

Tablets were prepared in the usual way with a fracture drill and the following composition:

EXAMPLE 7

Ampoule solutions of the following composition were prepared in the usual way:

Claims (9)

1. Process for the preparation of a tetrapeptide amide of formula wherein all the amino acids with an asymmetric center exhibit the L configuration, as well as acid addition salts thereof, characterized in that mana) converts the dipeptide of formula with a dipeptide of the general formula wherein R, represents hydroxy or a residue which activates the carboxyl group, or that manb) converts a tripeptide of the general formula in which R^ has the meaning given above, with the amide of formula or that manc) reacts the tripeptide of formula with in which R^ has the above meaning, or that mand) cleaves off the protecting group(s) of a tetrapeptide of the general formula wherein R» represents hydrogen or an amino protecting group, R3 hydrogen or a group protecting the imidazole function and R 4 hydrogen or an amide protecting group, whereby at least one of the residues R 2 / R 3 and R 4 is different or that mane) amidates a tetrapeptide of the general formula wherein X represents hydroxy, a residue that activates the carboxyl group or the residue -OR,-, wherein R^ means alkyl, aryl, aryl-lower alkyl or the residue of a polymeric support material, or that man-f) in a tetrapeptide of the general formula wherein R6 produces the similarly protected glutamine or glutamic acid residue, Y a residue which activates the carboxyl group, the residue -NHR^ , in which R^ has the above meaning, or the residue -OR^ , in which R^ has the above meaning, during simultaneous or previous cleavage of any protective group, the pyroglutamine ring ends and that, if desired, one transfers an obtained compound of formula I' in an acid addition salt, whereby all the amino acids with an asymmetric center in formulas II to X exhibit the L configuration. 2. Process for the production of pharmaceutical preparations, characterized by mixing the compound of formula in which all the amino acids with a center of asymmetry exhibits the L configuration, or a pharmaceutically usable acid addition salt thereof, as an active ingredient with suitable for therapeutic administration, non-toxic, inert, in and of themselves in such preparations common, solid or liquid carriers and/or excipients. 3. Pharmaceutical preparation, characterized in that it contains the compound of formula wherein all the amino acids with an asymmetric center exhibit the L configuration, or a pharmaceutically usable acid addition salt thereof, as well as a pharmaceutical carrier material. 4. The dipeptide of formula in which the proline exhibits the L configuration. 5. The tripeptide of formula in which the amino acids with the center of asymmetry exhibit The L configuration. 6. The tetrapeptide of the general formula | where R 2 represents hydrogen or an amino protecting group, hydrogen or a group protecting the imidazole function and R^ is hydrogen or an amide protecting group, whereby at least one of the residues R2/R3 and R^ is different from hydrogen and the amino acids with the center of asymmetry exhibit the L configuration. 7. Tetrapeptides of the general formula wherein X produces hydroxy, a residue that activates the carboxyl group or the residue -OR^ , wherein R^ produces lower alkyl, aryl, aryl-lower alkyl or the residue of a polymeric support material and the amino acids with an asymmetric center exhibit the L configuration. 8. Tetrapeptides of the general formula in which Rg means the optionally protected glutamine or glutamic acid residue, Y a residue that activates the carboxyl group, the residue -NHR^ , in which R^ means hydrogen or an amide protecting group, or the residue -OR^ , in which R^ means lower alkyl, aryl, aryl-lower alkyl or the residue of a polymeric carrier material and the amino acids with the eta asymmetry center exhibit the L configuration. 9. L-pyroglutamyl-L-histidyl-L-prolyl-(3-alanine amide) and its acid addition salts.