CH637626A5 - Process for the preparation of peptides - Google Patents

Description

The invention relates to a process for the preparation of peptides and their derivatives.

More specifically, the present invention relates to a process for the preparation of peptides and their derivatives which have a morphine-like effect. As is commonly believed and the term is used hereinafter, a morphine agonist is a compound whose biological activity mimics that of the natural alkaloid.

The pharmacological properties and therapeutic uses of morphine are well documented in the literature, see, for example, "The Pharmacological Basis of Therapeutics," edited by Goodman, L.S. and Gilman, A., published by The MacMillan Company, New York, 3rd Edition (1965), particularly Chapter 15, pages 247 to 266, and "Martingale: The Extra Pharmacopoeia", published by Blacow, NW, published by The Pharmaceutical Press, London, 26th edition (1972), in particular pages 1100 to 1106, to which the present text refers. However, as is well known (Goodman, LS et al., 1.c., Chapter 16), repeated administration of morphine to the patient can cause the drug to build up and get used to it, even leading to withdrawal symptoms. when the drug is stopped. For this reason, years of research have been carried out with the purpose of obtaining a compound which has the spectrum of action of morphine without its disadvantages.

The present new peptides of formula I produced according to the invention

R- (X1) m- (X2) n-X3-X4-X5-X6-R1 (I)

together with their salts, esters, amides, e.g. their N-alkylamides and N, N-dialkylamides, and their acid addition salts have the same effects as the morphine in both in vitro and in vivo tests.

In the compound of formula I, v

X1 and X2, which may be the same or different, each represent the residue of a basic O or L amino acid;

X3 is a D or L radical of the formula w1 y

R2- (CH_). (CHL.CH.CO-2 from where R2 is phenyl or 1,4-cyclohexadien-l-yl, a zero, 1 or 2, b zero or 1, one of the groups W and W1 the group - NR3- and the other is hydrogen, provided that W always means -NR3- when b is zero and that a is always 1 and b is always 1 and b is always zero when R2 is 1,4-cyclohexadiene-l- yl, in which R3 furthermore denotes hydrogen or alkyl, alkenyl, alkynyl, carboxyalkyl, carboxyalkenyl and carboxyalkynyl, and in which R2 is optionally by one or more of the groups hydroxy, alkoxy, alkanoyloxy, alkyl, nitro, trifluoromethyl, amino, N-

Alkylamino, halogen, N, N-dialkylamino or benzyloxy can be substituted, where in the latter group the Phe-15 nyl ring is optionally substituted by one or more of the groups hydroxy, alkoxy, alkanoyloxy, halogen, alkyl, nitro, trifluoromethyl, amino, N-alkylamino or N, N-dialkylamino can be substituted; further

X4 is a D radical from the group C-propargylglycyl, Ala-20 nyl, C-alkyl (2 to 4 carbon atoms) alanyl, valyl, norvalyl, leucyl, isoleucyl, norleucyl, prolyl, hydroxyprolyl, tryptophyl, asparaginyl and glutaminyl, where each of the radicals may optionally be Na-substituted with an alkyl group;

25 X5 glycyl, β-alanyl, C-methylalanyl or a D or L radical from the group C-propargylglycyl, alanyl, C-alkyl (2 to 4 C atoms) -anyl, valyl, norvalyl, leucyl, isoleucyl, Norleucyl, prolyl, hydroxyprolyl, tryptophyl, asparaginyl and glutaminyl, wherein each of the radicals may be Na-substituted with an Al-30 kyl group;

Xe glycyl or a D or L residue from the group methionyl, leucyl, isoleucyl, norleucyl, valyl, norvalyl, prolyl, hydroxyprolyl, alanyl and histidyl, and the definitions for X3;

35 R is hydrogen, aralkyl, alkyl, alkenyl, alkynyl, carboxyalkyl, carboxyalkenyl or carboxyalkynyl;

R1 is the hydroxyl of the 1-carboxyl group of the C-terminal amino acid residue or a group which replaces this 1-carboxyl group from the group -CH2OR4, in which 40 R4 is hydrogen or alkanoyl, and 5-tetrazolyl, which is optionally in 1- or The 2-position can be substituted by an alkyl or benzyl, the phenyl ring in the latter group optionally being substituted by one or more of the groups hydroxyl, alkoxy, alkanoyloxy, halogen, alkyl, nitro, trifluoromethyl, amino, N-alkylamino or N, N- Di-alkylamino can be substituted; and m and n represent zero or 1, with the exception of the amides, e.g. B. the N-alkylamides and N, N-dialkylamides, and their acid addition salts, wherein X6 is a D or L radical of the formula so

55 NO

1

wherein R3 is hydrogen or alkyl.

The abbreviations for 60 amino acids and their radicals used in the present text are known to the person skilled in the art and can be found, for example, in Biochemistry, edition 11, page 1726 (1972). In the present text, everything relates to the L-configuration of the chiral amino acids and their radicals, unless expressly stated otherwise.

The term "basic amino acid" used in the present case means an amino acid with two basic functions and a carboxyl group. So can

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4th

for example the residues X1 and X2 lysyl (D and L), homo-arginyl (D and L), ornithyl (D and L), histidyl (D and L), a, 6-diaminobutyryl (D and L) and arginyl (D and L) mean.

In the case of the compounds of the formula I, the alkyl substituents of the radicals X4 and X5 which are optionally present preferably have 1 to 4, preferably 1 to 2, carbon atoms. The alkyl portion of the alkylalanyl for X4 and X5 preferably has 1 to 2 carbon atoms. In the case of the phenyl or 1,4-cyclohexadien-l-yl ring R2 of the radical X3 and in the case of the benzyloxy substituents which may be present in this case, the halogen substituents which may be present can be fluorine, chlorine, bromine and iodine, and they contain any alkyl - And alkoxy substituents together with the alkyl moieties of the optionally present alkanoyloxy, N-alkylamino and N, N-dialkylamino substituents preferably 1 to 4, in the most favorable case 1 or 2, carbon atoms.

If the phenyl or cyclohexadien-l-yl ring of the radical X3 has 1 or 2 substituents, the groups are preferably in the 3- and / or 4-position of the ring.

If R or the group R3 of the radical X3 are alkyl, then this, together with the portion mentioned in the carboxyalkyl group, may in particular have 1 to 4 carbon atoms, for example 1 or 2 carbon atoms, but groups with, for example, 1 to 10 or 1 are also up to 20 carbon atoms included. Mean R and R3 e.g. Carboxyalkenyl or carboxyalkynyl or only alkenyl and alkynyl, these groups together with the stated proportions can preferably have 2 to 4 carbon atoms, but groups with, for example, 2 to 10 or 2 to 20 carbon atoms are also included in the formula I. For the alkenyl and alkynyl groups, the allyl (a, β or y) and propargyl can be regarded as particularly valuable.

If R is an aralkyl group, this can be, for example, a benzyl group which can optionally be substituted in the phenyl ring by one or more groups. These in turn can be the above-mentioned substituents for the benzyloxy group of group R2 in group X3.

If R1 is a group —CH2OR4, in which R4 is alkanoyl, the alkyl part of the alkanoyl group preferably has 1 to 4, at best 1 or 2, carbon atoms. If R1 is a 5-tetrazolyl group, the alkyl substituent optionally present preferably has 1 to 5 carbon atoms and the benzyl substituent optionally present can itself be substituted in the phenyl ring by one or more groups, these substituting the above-mentioned substituents for the benzyloxy group of group R2 correspond to X3 in the rest.

Among the esters of the peptides of formula I e.g. the alkyl esters and the aryl esters are understood. The alkyl esters include, in particular, the esters in which the alkyl group has 1 to 4 carbon atoms, for example the methyl, ethyl and t-butyl esters, but also the esters in which the alkyl group has 1 to 10 or 1 to 20 carbon atoms, for example has included. The aryl esters include e.g. B. the phenyl esters, e.g. B. the halophenyl esters, where the halogen e.g. Chlorine means, as in p-chlorophenyl ester.

In the case of the N-alkyl and N, N-dialkylamides of the peptides of the formula I, the alkyl groups can preferably have 1 to 5 carbon atoms, but alkyl groups with e.g. 1 to 10 or 1 to 20 carbon atoms included. In the case of the N, N-dialkylamides, the alkyl groups can be the same or different. In the present case, the amides of the peptides include not only the amides which, according to the concept, are derived from ammonia, but also the amides derived from heterocyclic bases, such as, for example, pyrrolidine, piperidine and morpholine, i.e. 5 z. B. the pyrrolidinamides, the piperidinamides and the morpholine amides.

A subgroup of the amides, the N-alkylamides and the N, N-dialkylamides of the peptides of the formula I, can be represented by the formula

10 c

R

r-Cx1) - (x2) -x3-x4-x5-x6-n ^ /

m n \

\ r6

wherein X1, X2,; X3, X4, X5, X6, m, n and R have the same meaning as in formula I, and wherein Rs and R6 together with the nitrogen to which they are attached are an amino, pyrrolidino, Piperidino, morpholino, N-alkylamino and N, N-dialkylamino group mean, the alkyl group in each case generally having 1 to 20 carbon atoms.

25 With the acid addition salts of the peptides of the formula I and their derivatives mentioned above, the effectiveness lies in the base and the acid is of less importance, although of course pharmacologically and pharmaceutically acceptable acids 30 are preferably important for the recipient for therapeutic purposes for the recipient. Examples of such suitable acids are a) mineral acids: e.g. Hydrochloric acid, hydrobromic acid, phosphoric acid, metaphosphoric acid, nitric acid and sulfuric acid;

35 b) Organic acids: e.g. Tartaric acid, acetic acid, citric acid, malic acid, lactic acid, fumaric acid, benzoic acid, glycolic acid, gluconic acid, gulonic acid, succinic acid and arylsulfonic acids, for example p-toluenesulfonic acid.

40 However, the pharmaceutically and pharmacologically acceptable acid addition salts together with the acids that are not so acceptable (e.g. salts of hydrofluoric acid and perchloric acids) are useful for the isolation and purification of the bases. Of course, the less acceptable salts are just as valuable for the preparation of the acceptable salts, which has long been known to those skilled in the art. The peptides and their derivatives, which have several free amino groups, can be obtained in the form of the mono- or poly-acid addition salts or as mixed salts of several acids. Similarly, in the case of the salts of the peptides (the peptide being present as a carboxylate anion together with a cation), the importance of the cation is less important, although for therapeutic purposes it should of course preferably be pharmacologically and pharmaceutically acceptable to the recipient. Examples of these suitable cations include Sodium and potassium.

The morphine-like properties of the peptides of the formula I and their derivatives described above are described below using a few examples.

60

A) In vitro

1. Inhibition of the neural induced contractions of the isolated vas deferens of the mouse, tested according to the method of Hughes et al. (Brain Research, Vol. 88 (1975), 65 page 296) (using current surges at 0.1 Hz), the inhibition by the known narcotic antagonist naloxone (IN-allyl-7,8-dihydro-14-hydroxynormorphinone ) was repealed.

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2. Reduction of the electrically induced contractions of the isolated guinea pig intestine after preparation for stimulation by the method of Paton (Brit. J. Pharmacol., Volume 12 (1957), pages 119 to 127). (Each segment of the intestine was fitted with an anode and hung with a weight of 2 to 3 g.

Stimulus characteristics: frequency: 0.1 Hz; Duration: 0.4 minutes; Voltage (supramaximal): 30 to 40 V; the contractions were transduced isotonic.

B) In vivo

1. The compounds have an analgesic effect, for example they are effective in mice in the known standard method of the "hot plate" test, which is a modification of the method according to N.B. Eddy, et al. (J. Pharm. Exp. Therap., Volume 107, (1953), page 385). The compounds were administered by intracerebroventricular injection and the effect was eliminated by «naloxone».

2. The compounds also act against cough, for example when testing guinea pigs using the method of Boura et al., Brit. J. Pharmacol., Vol. 39 (1970), page 225.

3. The compounds act against diarrhea, for example they effectively reduce the diarrhea caused by castor oil in rats.

The following compounds can be mentioned as subclasses of the peptides of the formula I and their derivatives, in which

1. m and n both zero;

2. R is hydrogen;

3. X3 L-tyrosyl;

4. X4 D-alanyl;

5. X5 glycyl;

6. X6 L-phenylalanyl, L-4-chlorophenylalanyl and L-4-nitrophenylalanyl, preferably L-4-chlorophenylalanyl and L-4-nitrophenylalanyl, in the most favorable case L-4-nitro-phenylalanyl, and

7. R1 is the hydroxyl of the 1-carboxyl group of the C-terminal amino acid residue.

As further subclasses of the peptides of the formula I and their derivatives, the compounds of the formula

H • Tyr • D-Ala • Gly • Xe • R1,

wherein

X6 L-phenylalanyl, L-4-chlorophenylalanyl and L-4-nitrophenylalanyl, and

R1 is the hydroxyl of the 1-carboxyl group of the C-terminal amino acid residue.

Preferred esters of this subclass are the alkyl esters in which the alkyl group has 1 to 10, preferably 1 to 4, in the most favorable case 1 or 2, carbon atoms and the preferred N-alkylamides and N, N-dialkylamides are groups in which the alkyl groups 1 to 5 , preferably 1 or 2, have carbon atoms.

The peptides of the formula I and their derivatives mentioned above can be prepared in the ways already known for the preparation of compounds of analogous structure. They can be produced by successive coupling of the corresponding amino acids using the classic methods of peptide synthesis or by solid-phase methods, or else by initial production and subsequent coupling of the peptide subunits.

These reactions can either be e.g. B. by activating the carboxyl group, the incoming amino acid and by protecting the non-reacting amino and car-boxyl groups. These are standard procedures in peptide production. Details of suitable activating and protective (masking) groups and suitable reaction conditions (both for the coupling reactions and for the removal of the protective groups), with the minimum of reactivation occurring, can be found in the literature listed below, which serves only for explanation.

a) GB Patent 1,042,487; 1,048,086 and 1,281,383.

b) Schröder and Lüebke, "The Peptides" (Academic Press) (1965).

c) Bellean and Malek, J. Am. Chem. Soc., Vol. 90, (1968), page 165.

d) Tilak, Tetrahedron Letters, (1970), page 849.

e) Beyerman, Helv. Chim. Acta., Vol. 56, (1973), page 1729.

f) Stewart and Young, "Solid Phase Peptide Synthesis" (W.H. Freeman and Co.) (1969).

Depending on the reaction conditions, the peptides of the formula I and their derivatives can be obtained either in the form of the free base or as their acid addition salt or (in the case of the peptide itself) as a salt. The acid addition salts can be converted to the free bases or salts of other acids and the bases can in turn be converted to their acid addition salts, in a manner known to those skilled in the art. In a similar manner, the peptides can be converted into their salts and their salts into the peptides or into other salts by the known methods.

The peptides of formula I and their derivatives mentioned in claim 1 and their acid addition salts are inventively by condensing a compound of formula II

R-Y'-OH (II),

wherein R has the meanings given in formula I and Y1 means a partial sequence identical to the corresponding N-terminal partial sequence in formula I, with a compound of formula III

H-Y2-R! (III),

in which R1 has the meanings given in formula I and Y2 represents the addition of the product defined above, forming a carboxamide bond, it being possible for the compounds of the formulas II and III to be protected and / or activated and the protection of the groups is subsequently removed and the reaction product is converted into the base or into a salt or into one of its acid addition salts.

It is obvious to a person skilled in the art that the arginyl (D or L) and homoarginyl (Har) (D or L) radicals can not only be inserted into the peptide chain in the manner described above, but also in situ in the resulting chain can be formed or in one of its subunits by guanidation of an ornithyl (D or L) or lysyl (D or L) residue using a reagent such as l-guanyl-3,5-dimethylpyrazole, can be formed.

It is also evident that other in situ conversions of the peptides of formula I and their derivatives are possible. So the amides, e.g. B. the N-alkylamides and the N, N-dialkylamides, for example by reacting a peptide-alkyl ester, such as e.g. of the methyl ester, with ammonia, a heterocyclic base or a mono- or dialkylamine as required. The peptide esters can be prepared from the peptides by standard esterifications and the esters can be made into the peptides

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Saponification. Hydroxy substituents in group R2 of group X3 can be substituted in alkoxy or benzyloxy groups by using the appropriate diazoalkane, e.g. Diazomethane can be converted to give methoxy groups. Likewise, benzyloxy and alkanoyloxy substituents in group R2 of group X3 can be removed leaving hydroxy groups by hydrogenolysis in methanol using 10% palladium-on-carbon as a catalyst or by alkaline hydrolysis. These hydroxy groups can in turn be converted to alkanoyloxy groups using the conventional alkanoylation processes. All of these known methods in peptide chemistry are listed with reference to the literature cited, from which further details of the reaction conditions and the corresponding protective methods or removal of the protection can be found.

Because of their morphine activity, the peptides of formula I together with their pharmacologically and pharmaceutically acceptable salts, esters, amides, e.g. with the N-alkylamides and N, N-dialkylamides, as well as their pharmacologically and pharmaceutically acceptable acid addition salts for the treatment of mammals in both human and veterinary medicine in all cases where an agent with a morphine-like effect is indicated. Here are e.g. the following possible uses are shown:

1. Pain relief (analgesia), e.g. for pain due to smooth muscle spasms such as kidney or biliary collic, pain for incurable diseases such as for cancer, pain in the postoperative period and labor pains.

2. calming, e.g. for pre-anesthetic medication, tranquillization, for falling asleep, especially if the insomnia is due to pain or cough, as well as to relieve general anxiety.

3. Stopping cough.

4. Elimination of breathing disorders, e.g. in acute failure of the left ventricle or in pulmonary edema.

5.Calling a constipation, e.g. after an ileostomy or colostomy, and in the treatment of diarrhea and dysentery.

6. Arousing euphoria and treating depression, e.g. B. associated with pain relief for incurable diseases such as in cancer.

For each area of application, the required amount of the peptide or its derivative or acid addition salt (hereinafter referred to as active ingredient) varies depending on the mode of administration and depends on the severity of the clinical picture to be treated and is ultimately determined by the doctor or veterinarian. In general, however, the dosage for the abovementioned applications is in the range from 0.0025 pg to 40 mg / kg body weight of the mammal, preferably 0.025 pg to 4.0 mg / kg, at best 0.25 to 400 pg / kg body weight ( where all dosages are calculated with respect to the peptide base).

The active ingredient can be administered in any suitable manner depending on the condition to be treated, and suitable uses can be oral, rectal, nasal, local (buccal), vaginal and parenteral (including subcutaneous, intramuscular and intravenous administration). It is obvious that the preferred mode of administration varies depending on the condition to be treated, e.g. administration into the spinal cord can be beneficial for the elimination of labor pains.

Although it is possible to administer the active ingredient as a raw chemical, it is advantageous to present it in a pharmaceutical preparation.

The present preparations, both for veterinary and for human medicine, contain the active substance defined above together with one or more acceptable carriers and, if appropriate, together with other therapeutic active substances. The carrier (s) must be acceptable in the sense that they are compatible with other components of the preparation and are not harmful to the recipient. It is advantageous if the preparations do not contain any oxidizing agents and other substances with which the peptides are known to be incompatible.

The preparations can be for oral, rectal, nasal, local (buccal), vaginal or parenteral (including subcutaneous, intramuscular and intravenous) administration, although the most suitable administration form always depends on the active ingredient and the condition to be treated. The formulations can usually be in single-dose formulations and can be prepared in the manner known in pharmacy. In all forms of preparation, the active ingredient is brought together with the carrier, the latter being able to consist of one or more auxiliaries. In general, the preparations are prepared in such a way that the active ingredient is thoroughly mixed with liquid carriers or finely comminuted solid carriers or with both, and then the product is brought into the desired shape as required.

The present preparations for oral administration can be in divided units, such as capsules, cachets or tablets, each unit containing a predetermined amount of the active ingredient. Furthermore, they can be present as powder or granules or as a solution or suspension in an aqueous or non-aqueous liquid or as a liquid oil-in-water or water-in-oil emulsion. The active ingredient can also be in the form of a bolus, electuary or paste.

A tablet can be produced by pressing or molding, optionally with one or more auxiliary substances. Pressed tablets can be produced by pressing in a suitable device, the active ingredient being in free-flowing form, for example in the form of powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface-active agent or dispersant. Molded tablets can be made by molding in a suitable device, moistening a mixture of the powdered compound with an inert liquid diluent.

Preparations for rectal administration can be used as suppositories together with the usual carrier, e.g. Cocoa butter are present, whereas a suitable preparation for nasal administration is nasal drops which contain the active ingredient in an aqueous or oily solution.

Preparations which are suitable for local administration in the mouth are e.g. Lozenges contain the active ingredient in a flavor base, usually sucrose and gum arabic or tragacanth. Pastilles contain the active ingredient in an inert base, such as gelatin and glycerin or in sucrose and gum arabic.

Preparations for vaginal administration can be in the form of pessaries, creams, pastes or sprays, which in addition to the active ingredient also contain the corresponding known carriers.

Preparations for parenteral administration usually contain sterile aqueous solutions of the active ingredient, these solutions preferably containing the blood of Emp6

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are isotonic. These preparations are usually prepared by dissolving the solid active ingredient in water into an aqueous solution and making this solution isotonic with the blood of the recipient and sterilized. The preparations can be in single dose or multiple dose containers, e.g. B. in sealed ampoules or glasses.

Preparations which are suitable for nasal administration and in which the carrier is a solid contain a coarse powder with a particle size of, for example, 20 to 500 microns, which is used similarly to snuff, i.e. by rapid inhalation through the nose from a container that is held close to the nose.

It goes without saying that, in addition to the above-mentioned components of the present preparations, other components may also be present, such as e.g. Diluents, buffers, flavoring agents, binders, surfactants, thickeners, lubricants, preservatives (including antioxidants) and the like.

If the preparation is in single-dose form, for example in one of the abovementioned forms, for both human and veterinary use, each individual unit usually contains the active ingredient in an amount of 0.125 (ig to 2 g, preferably 1) , 25 jxg to 200 mg, at best 12.5 [ig to 20 mg, all weights being calculated on the peptide base.

Exemplary embodiments of the present invention are described in more detail below with the aid of the examples. All temperatures are in ° C.

tries

The following abbreviations have been used below:

HOBT 1-Hydroxybenzotriazole DCCI Dicyclohexylcarbodiimid DMF Dimethylformamide BOC Tertiary Butyloxycarbonyl BZL Benzyl.

The peptides were checked by thin layer chromatography on «Merck» silica gel plates using the following solvent systems:

1. Methyl ethyl ketone

2. n-butanol: acetic acid: water (3: 1: 1)

3. Chloroform: methanol: 32% acetic acid (120: 90: 40)

4. Chloroform: methanol: 32% ammonia (120: 90: 40)

5. n-Butanol: acetic acid: ethyl acetate: water

(1: 1: 1: 1)

6. Chloroform: methanol (8: 1)

7. Chloroform: methanol: 32% acetic acid (120: 90: 5)

8. Chloroform: methanol: 32% ammonia (120: 90: 5)

The optical rotations were determined using an automatic “Bendix NPL” polarimeter.

The contents of individual amino acids in the peptide hydrolyzates (6n • HCl at 110 ° C. for 24 hours in evacuated sealed test tubes) were determined using a “Beckman Spinco” amino acid analyzer (model 120C) or using a “Rank Chromostak” amino acid analyzer certainly.

The following general procedures were used during the synthesis of the peptides:

a) The couplings were carried out in DMF and mediated by DCCI.

b) The amino acid ester hydrochlorides were converted into the free esters by adding a tertiary base, either of triethylamine or N-methylmorpholine.

c) During the coupling stage, HOBT was added as soon as the fragment condensation included a peptide with an optically active C-terminal amino acid.

d) The couplings were carried out in a cold room at +4 ° C for 24 hours.

e) After coupling, the cleaning was carried out by washing with acid and base in order to remove unreacted reactants.

f) Alkaline saponifications were carried out in aqueous methanol using an autotitrator at pH 11.5 to 12.0 using n • NaOH.

g) The benzyloxycarbonyl protecting groups were removed by hydrogenolysis in methanol / acetic acid with 10% palladium on carbon.

h) The resulting acetates from the above hydrogenolysis were converted into the corresponding hydrochlorides by adding methanolic hydrogen chloride.

i) The benzyl protecting groups were removed by hydrolysis in methanol with 10% palladium on carbon.

j) Tertiary butyl and tertiary butyloxycarbonyl protective groups were removed with n-HCl in acetic acid in the presence of anisole as a rinsing agent. The split lasted 60 to 90 minutes.

The final peptides were isolated as their hydrochlorides and lyophilized from aqueous solution.

Example 1 H • Tyr • D-Ala • Gly • Phe • OH This substance was prepared according to the scheme in Table 1. As the hydrochloride addition salt after the lyophilization from aqueous solution, the reaction product had the following characteristic values:

Rf: 0.412; 0.664; 0.238 [a] D25: + 64.6 ° (c = 0.5 in methanol).

Example 2 H • Tyr • D-Ala • Gly • Phe • OMe This substance was prepared from the reaction product of Example 1 by reaction with methanolic hydrogen chloride. As the hydrochloride addition salt after the lyophilization from aqueous solution, the ester had the following characteristic values:

Rf: 0.572; 0.883; 0.635 [a] D25: + 57.2 ° (c = 0.5 in methanol).

The same reaction product with identical characteristic values resulted from the treatment of the reaction product of process stage 5 in Table 1 in the following way:

Bzl

BOC.

Tyr.

D-

Ala.

Gly.

Phe.

7

H

O

Pd / C

H2

BOC.

Tyr.

D-

■ Ala.

Gly.

Phe.

n-HCl / HOAc

W

H. Tyr. D-Ala. Gly. Phe. OMe s

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Table 1

H. Tyr

D. Ala

BOC-D-Ala.OH

Bzl I

BOC-Tyr.OH

BOC-D-Ala

H.D-Ala

Gly Phe.OH

H.Gly.OMe HOBT / DCCI coupling: Gly.OMe n.HCl / HOAc Gly.OMe

@

Bzl I

BOC-Tyr

Bzl l

BOC-Tyr

Bzl I

BOC.Tyr Bzl

I.

BOC.Tyr BOC.Tyr H.Tyr

HOBT / DCCI coupling

D-Ala

©

n.NaOH / MeOH D-Ala

Gly.OMe

Gly.OH

D-Ala

D-Ala

D-Ala

D-Ala

Gly

Gly

Gly

Gly

H. Phe.OMe

HOBT / DCCI coupling - Phe.OMe n.NaOH / MeOH - Phe.OH

10% Pd / C H,

Phe.OH

n.HCl / HOAc - Phe.OH

The following peptides with the correspondingly shown terminal derivatives were characterized in the usual manner.

drive the peptide chemistry analogously to the method described in the previous 50 -OMe: methyl ester examples. On the C- -OHeptyl (n): n-heptyl esters

-NHEt: ethyl amide.

Example No. Connection

Rf

[a] D25 (in methanol)

3rd

H

• Tyr '

D-Ala • Gly • D-Phe • OH

0.605; 0.447

4th

H

Tyr '

D-Ala • Gly-Phe (4-Cl) -OH

0.417

+ 55.9 ° (c =

0.3)

5

H

• Tyr '

■ D-Ala • D-ala • Phe • OH HCl

0.723; 0.675

6

H

■ Tyr • D-Ala • Gly • Phe ■ OHeptyl (n) HCl

0.652

+ 46.1 ° (c =

0.2)

7

H

• Tyr '

D-Ala • Gly • Phe (4-N02) • OMe HCl

0.607; 0.548

+ 59.4 ° (c =

0.5)

8th

H'

■ Tyr

D-Ala • Gly • Phe (4-N02) • NHEt HCl

0.532; 0.687; 0.618

+ 36.9 ° (c =

0.5)

9

H

■ Tyr •

D-Ala • Gly • Phe (4-N02) • OH HCl

0.432; 0.497; 0.558

+ 66.3 ° (c =

0.5)

Example: Pharmacological Action The peptides of the previous examples were tested for the following effects according to standard pharmacological procedures.

A) Analgesia in mice with the hotplate test (modification of the method according to N.B. Eddy, et al., J. Pharm. Exp. Therap., Volume 107 (1953), page 385, the peptide being administered by intracerebroventricular injection).

637 626

B) Effect against diarrhea in rats. In this procedure, the rats were left to starve for 24 hours, the peptide was then administered subcutaneously and, after a further 15 minutes, the rats were given 1 ml of castor oil orally.

The respective ED50 values were calculated from the values obtained (i.e. the required dose, which produces the corresponding effect in 50% of the animals), expressed by the weight of the peptide base per mouse / or rat.

peptide

analgesia

Effect against from example no.

(Mouse: hot plate)

Diarrhea (rat)

EDS0 i.c.v.

EDso s.c.

((ig / mouse)

(mg / kg rat)

1

0.7

5.0

2nd

0.2

0.7

4th

5.0

1.0

6

3.0

3.0

7

0.5

0.3

8th

0.07

0.3

9

1.0

0.5

B) suppositories (5 mg / suppository) compound of the formula I suppository mass (Massa Esterinum C)

10th

Pharmaceutical preparation A) Tablet preparation (20 mg / tablet)

Compound of formula I 20 mg

Milk sugar 76 mg

Corn starch 10 mg

Gelatin 2 mg

Magnesium stearate 2 mg

The compound of formula I, the milk sugar and the corn starch are mixed together. The granulate is then produced with the gelatin dissolved in water. After the granules have dried, the magnesium stearate is added and the whole is compressed into tablets of 110 mg.

The suppository mass is melted at 40 ° C. Then the compound of formula I is gradually incorporated into finely powdered form and mixed until homogeneous. The mass is then poured into suitable molds, 2 g per suppository mold, and left to stand until solid.

Massa Esterinum C is a commercially available suppository mass, which consists of a mixture of mono-, di- and triglycerides of saturated vegetable fatty acids. It is distributed by Henkel International, Düsseldorf.

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250 mg 100 g

C) Pessary (5 mg / product)

Compound of formula I 5 mg

Milk sugar 400 mg i5 "Povidone" poly- (l-vinyl-2-pyrrolidinone) 5 mg

Magnesium stearate 5 mg

The compound of formula I and the milk sugar are mixed together. Then it is granulated with a 50% “povidone” solution in aqueous ethanol. After the granules have dried, the magnesium stearate is added and the whole is pressed into pessaries of 415 mg each with appropriately shaped punches.

D) Freeze-dried injection (100 mg / ampoule)

25 compound of formula I 100 mg

Water ad injection ad 2.0 ml

The compound of formula I is dissolved in the water. The solution is sterilized by passing through a membrane filter with a pore size of 0.2 μm, after which the filtrate is collected in a sterile container. Then, under aseptic conditions, 2 ml per ampoule are filled into sterile glass ampoules and freeze-dried. The ampoules are closed with sterile rubber closures and secured with an aluminum closure 35.

The injection is restored before use by adding an appropriate volume of water for injections or with sterile saline.

In the examples given above, the weight of the compound of the formula I is calculated in each case on the peptide base.

s

Claims (14)

637 626 1. Process for the preparation of peptides of the formula I. R- (X1) m- (X2) n-X3-X4-X5-X6-R1 (I), wherein X1 and X2, which may be the same or different, each represent the residue of a basic D or L amino acid; X3 is a D or L radical of the formula w1 y r2- (ch “). (Ìh). .ch.co- 2. The method according to claim 1 for the preparation of an amide, a compound of formula I, characterized in that the correspondingly obtained alkyl ester compound of formula I is allowed to react with ammonia, a heterocyclic base or a mono- or dialkylamine. 2 from which R2 is phenyl or 1,4-cyclohexadien-l-yl, a zero, 1 or 2, b zero or 1, one of the groups W and W1 is the group -NR3- and the other is hydrogen, provided that W is always -NR3- means when b is zero and that a is always 1 and b is always zero when R2 is 1,4-cyclohexadien-l-yl, furthermore R3 is hydrogen or alkyl, alkenyl, alkynyl, carboxyalkyl, carboxyalkenyl and carboxyalkynyl, and in which R2 can be optionally substituted by one or more of the groups hydroxy, alkoxy, alkanoyloxy, alkyl, nitro, trifluoromethyl, amino, N-alkylamino, halogen, N, N-dialkylamino or benzyloxy, the latter group being the Phenyl ring can optionally be substituted by one or more of the groups hydroxyl, alkoxy, alkanoyloxy, halogen, alkyl, nitro, trifluoromethyl, amino, N-alkylamino or N, N-dialkylamino; further X4 is a D radical from the group C-propargylglycyl, alanyl, C-alkyl (2 to 4 carbon atoms) alanyl, valyl, norvalyl, leucyl, isoleucyl, norleucyl, prolyl, hydroxyprolyl, tryptophyl, asparaginyl and glutaminyl, wherein each of the radicals may optionally be Na-substituted with an alkyl group; X5 glycyl, ß-alanyl, C-methylalanyl or a D or L radical from the group C-propargylglycyl, alanyl, C-alkyl (2 to 4 carbon atoms) - alanyl, valyl, norvalyl, leucyl, isoleucyl, norleucyl , Prolyl, hydroxyprolyl, tryptophyl, asparaginyl and glutaminyl, wherein each of the radicals may be Na-substituted with an alkyl group; X6 glycyl or a D or L radical from the group methionyl, leucyl, isoleucyl, norleucyl, valyl, norvalyl, prolyl, hydroxyprolyl, alanyl and histidyl, and the definitions for X3; R is hydrogen, aralkyl, alkyl, alkenyl, alkynyl, carboxyalkyl, carboxyalkenyl or carboxyalkynyl; R1 is the hydroxyl of the 1-carboxyl group of the C-terminal amino acid residue or a group which replaces this 1-carboxyl group from the group -CH2OR4, in which R4 is hydrogen or alkanoyl, and 5-tetrazolyl, which is optionally in 1- or 2 -SteIlung can be substituted by an alkyl or benzyl, in the latter group the phenyl ring optionally by one or more of the groups hydroxy, alkoxy, alkanoyloxy, halogen, alkyl, nitro, trifluoromethyl, amino, N-alkylamino or N, N-di- alkylamino can be substituted; and m and n zero or 1 mean, or their salts, esters, amides or their acid addition salts, with the exception of the amides and their acid addition salts, in which Xe is a D or L radical of the formula CH ".CH.CO- NR I wherein R3 is hydrogen or alkyl, characterized in that a compound of formula II R-Y1-OH (II), wherein R has the meanings given in formula I and Y1 means a partial sequence identical to the corresponding N-terminal partial sequence in formula I, with a compound of formula III H-Y2-R1 (III), wherein R1 has the meanings given in formula I and Y2 represents the supplement to the product described above, and wherein the compounds of the formulas II and III can, if appropriate, be protected and / or activated, condensed to form a carboxamide bond and then this protection is canceled and the product obtained is optionally converted into the base, a salt or an acid addition salt. 2nd PATENT CLAIMS 3,637,626 3. The method according to claim 2 for the preparation of an N-alkyl amide or an N, N-dialkyl amide of a compound of formula I. 4. The method according to claim 1 for the preparation of an ester of a compound of formula I, characterized in that a correspondingly obtained compound of formula I, wherein R1 represents the hydroxyl group of the 1-carboxyl group of the C-terminal amino acid residue, is esterified. 5 5. The method according to claim 1 for the preparation of a compound of formula I, in which R1 represents the hydroxyl group of the 1-carboxyl group of the C-terminal amino acid residue, characterized in that the correspondingly obtained ester compound of formula I is saponified. 6. The method according to any one of claims 1 to 5, characterized in that one uses a compound of formula II, wherein m and n each represent zero. 7. The method according to any one of claims 1 to 6, characterized in that one uses a compound of formula II, wherein R is hydrogen. 8. The method according to any one of claims 1 to 7, characterized in that one uses compounds of formula II or III, wherein X3 is L-tyrosyl. 9. The method according to any one of claims 1 to 8, characterized in that one uses compounds of formula II or III, wherein X4 is D-alanyl. 10th 15 20th 25th 30th 35 40 45 50 55 60 65 wherein X6 L-phenylalanyl, L-4-chlorophenylalanyl or L-4-nitrophenylalanyl; and R1 is the hydroxyl of the 1-carboxyl group of the C-terminal amino acid residue. 10. The method according to any one of claims 1 to 9, characterized in that one uses compounds of formula II or III, wherein X5 is glycyl. 11. The method according to any one of claims 1 to 10, characterized in that one uses a compound of formula III, wherein X6 is L-phenylalanyl, L-4-chlorophenylalanyl or L-4-nitrophenylaIanyl. 12. The method according to any one of claims 1,4 and 6 to II, characterized in that one uses a compound of formula III, wherein R1 is the hydroxyl of the 1-carb-oxyl group of the C-terminal amino acid residue. 13. The method according to claim 1, characterized in that the compounds of the formulas II and III are chosen such that they end product is a peptide or one of its derivatives of the formula mentioned in claim 1 H • Tyr • D-Ala • Gly • X6 • R1 14. The method according to any one of claims 1 to 13, characterized in that the pharmacologically and pharmaceutically acceptable salts or acid addition salts of the reaction product are prepared.