DD202693A5 - METHOD FOR THE PREPARATION OF A PEPTIDE - Google Patents

Abstract

The invention relates to a process for the preparation of a peptide with anorexigenic activity, inhibitory activity against excess gastric and pancreatic secretion and activating action on the central nervous system for the treatment of, for example, obesity, gastric and duodenal ulcers, disorders of consciousness and the like. The aim of the invention is to provide compounds with improved activity and long-lasting effect. According to the invention, peptides of the general formula A-B-C and pharmacologically acceptable salts thereof are provided. A is selected from the group L-pyroglutamyl, D-pyroglutamyl and L-homopyroglutamyl, B from the group L-histidyl, L-3'-methylhistidyl, D-histidyl, L-phenylalanyl, Lp-aminophenylalanyl and 1-beta- (pyrazolyl 1) -alanyl and C from the group glycine and lower alkyl esters thereof, glycinamide and lower alkylamides thereof, 2-amino-1-hydroxyethyl, D-alanine, L-beta- (2-thienyl) alanine and NHR high 1 are selected in which R is 1-lower-alkyl, with the proviso that C is not glycine or glycinamide, when A is L-pyroglutamyl and B is L-histidyl.

Description

'Berlin, 23.3.1982

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Process for the preparation of a peptide

Field of application of the invention

The invention relates to peptides of the general formula I.

A - B - G. (I)

and their pharmacologically acceptable salts.

The invention further relates to a process for the preparation of the compounds of the general formula I, intermediates used in this process, the use of these compounds as anorexigenic agents for the treatment of fatigue and other pathological conditions in which a reduction in feed or feed intake is indicated In mammals, as well as the use of these compounds for the treatment of pathological conditions which result in excess secretion of gastric acid and pancreatic fluid, such as gastric and duodenal ulcers or acute pancreatitis, and in the treatment of certain critical states of the central nervous system, such as disorders of consciousness or coma due to brain injury.

Characteristic of the known technical solutions

The abbreviations used for the amino acids and their residues as well as for the protecting groups are based on recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature (see Biochemistry, Vol. 11 (1972), p. (1972), S, 1726. Examples of these are:

(Pyro) -Glu = 5-oxoproline or pyroglutamic acidj homo- (pyro) -Glu - homo pyroglutamic acid j His = histidine ^ N -3-His 3 ¹ - »

I. line , i \ " _Λ

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Methylhistidine} Phe = phenylalanine p -HHgPhe = p-aminophenylalanine, β- (pyrazolyl-1) Ala = β- (pyrazol-1-yl) alanine} Gly s Glycine; Gly-ol 2-aminoethanol; and thi a β- (2-thienyl) alanine «

All amino acids have the natural or L-configuration, unless stated otherwise. "D-His is the residue of D-histidine, D- (pyro) -Glu is the residue of D-pyroglutamic acid and D-Ala is the residue of D-alanine ₀ The abbreviations Me and Bt are used for methyl and ethyl For protective groups, for example, the following abbreviations are used;

Boc = tert-butyloxycarbonyl} Z »benzyloxycarbonyl! Tos a tosyl} Dnp = dinitrophenyl} N = imidazole nitrogen of histidine} and Y »a suitable anchoring _s bound to a solid resin support used in the pest phase synthesis group, preferably

-0-GH ₂ -

The humurale appetite control, particularly over the Hypothalagus will be discussed for many years, see _e example, the review article by Schally et al _e, On _e I ₀ Med "Be ·, Vol. 248 (1964), p 79 and the references cited therein Since that time it has been known that the hypothalamus is involved in the regulation of food intake. Electrical stimulation of the lateral hypothalamic area leads to hyperphagia and its destruction to aphagia.

In contrast, stimulation of the ventromedial nucleus causes a reduction in nutritional intake and its destruction of hyperphagia and obesity *

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The obesity syndrome caused by the administration of gold thioglucose is partly the result of hyperphagia associated with hypothalamic lesions caused by this remedy. "Theories of neutral control of hunger, appetite, and satiety have been discussed by several authors. Various mechanisms have been proposed:

1) Thermostatic theory

According to this theory, a special dynamic effect of food and its influence on body temperature regulate the appetite »

2) Ohemostatic theories

These theories postulate that the appetite is regulated by intracellular or extracellular concentrations of glucose (glucostatic theory), lipids (lipostatic theory), and various metabolites, such as serum amino acids. "

3) Another group of theories assumes that digestive tract sensations associated with eating and in the presence of food in the stomach and intestine regulate the appetite "Among the factors involved in the development of satiety or the cessation of food intake Gastric contractions can be related to hunger, but these contractions are the same in animals that have been artificially rendered hypothalamic to an aphagic or hyperphagic state in animals that are deprived of food and water »

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These theories are not capable of explaining all the experimental findings "It may be that diabetic animals may feel hungry © Schally et al., Op. Cit., Suppose that obesity occurring in animals with hypothalamic lesions is due to a humoral rather than a humoral condition neurogenic mechanism and that the hypothalamus may develop a substance involved in central appetite control. Preliminary findings are presented indicating that the neurohypophysial extract contains a substance that influences the dynamic phase of weight gain in gold thioglycosis-treated mice. «

Further, a recent review article by Schally et al. On the status of research on the hypo thalamic control of feed intake and obesity, cf. Recent Progress in Hormone Research, Proceedings of the 1967 Laurentian Hormone Conference, Vol. 24 (1968), p. 497, especially S, 570 to 571 and the references cited therein. These references include the work of Schally u. Al. Science, Vol. 157 (1967) »S, 210, which shows that administration of dog duodenum-purified Enterogastrone reduces food intake for 15 hours without feed.» This effect is greatest during the first 30 minutes, but lasts the cumulative reduction at least 4 hours. Other peptides isolated from dog duodenum or colonic as well as glucagon, secretion, glucose and bovine serum albumin were found to be ineffective. These authors suppose that these effects are due to a direct elimination of starvation-gastric contractions or that they are released by the central nervous system with the release of

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Hypothalamic substances, although positive evidence for hypothalamic neurofluoreses responsible for indirect or direct control of appetite could not be

It appears that this positive evidence has been provided by Trygstad et al., Acta Endocrinologica, Bd. 89 (1978), p. 196. These authors isolated a number of peptides from the urine of patients congenital, more general Lipodystrophy (hypothalamus syndrome) suffered "Biese peptides caused corresponding metabolic effects. Nervous Anorexia Associates with Hypothalamic Oranges »In primary, nervous hypothalamic anorexia, the hypothalamic-pituitary axis is disturbed resulting in low gonadotropin release, amenorrhea, a loss of the daily rhythm of ACTH excretion, decreased thyrotropin excretion, and an initial increase and later decrease in somatotropin excretion "

Precipitation from urine samples from 25 patients with nervous anorexia was chromatographed on columns packed with Sephadex G-25 gel. A classification into four different chromatographic patterns was found. One was similar to the pattern of normal controls, one similar to that of schizophrenia patients 5 patients with hysteriform neurosis had a third pattern, and 10 girls with a primary "hypothalamic" type of nervous anorexia also showed typical chromatograms. "Fractions affecting mouse appetite were found only in the last group

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found. Two appetite-affecting paptides were isolated over several chromatographic stages. These peptides were injected into mice, producing an increased appetite, while the second caused an appetite reduction. The peptides are encased in peptide carrier proteins and thus sliced against enzymatic degradation, resulting in their isolation from the protein Urine allows »

The structure of the anorexigenic peptide was elucidated by Reichelt et al., Neuroscience, Bd, 3 (1978), S, 1207 in close collaboration with Trygstad. "It is the tripeptide H (pyro) -Glu" -His-GlyOH, which was confirmed by synthesis.

With an injection of 12 nM a day of appetizing peptides into mice over 20 days, feed intake decreased from 5.7 to 3 ₉ o g per day for about 6 months * body weight fell from an average of 35 g to a minimum of 24 G.

The tripeptide has no acute effect on blood glucose levels and serum insulin levels. Obviously it acts on receptors in the appetite controlling hypothalamic centers. "

The appetite-stimulating peptide increased daily food intake to over 10 g, with body weight rising to an average of 57 g. The structure of the appetite-stimulating peptide could not be identified «

Two similar factors showing increased and decreased feed intake behavior have also been reported in patients with genetic, metabolic obesity

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isolated _Φ

Object of the invention

The aim of the invention is to provide new peptides which have a long-lasting activity in terms of a reduction in appetite and cause a reduction in food intake,

Explanation of the essence of the invention

The invention has for its object to find peptides with the desired properties and processes for their preparation.

According to the invention, peptides of the formula ABC are prepared, A is a radical from the group L-pyroglutamyl (LH (pyro) -Glu), D-pyroglutamyl (DH (pyro) -Glu) and L-homopyroglutamyl (LH-homo- (pyro ) -Glu) means

B is a group selected from L-histidyl (L-His), D-histidyl (D-His), L-3 ¹ -methylhistidyl (LN ⁱⁿ 3-MeHis), L-phenylalanyl (L-Phe), Lp Aminophenylalanyl (Lp-NHgPhe) and L-.beta .- (pyrazolyl-1) -alanyl (L-.beta .- (pyrazolyl-1) Ala) and

C is a radical from the group glycine and its lower allyl ester (Gly-OR, where R is H or lower-allyl), glycine amide and its lower-alkylamides (Gly-NHR, where R is H or lower-alkyl), 2-amino 1-hydroxyethyl (glycol), D-alanine (D-Ala), L-β- (2-thienyl) alanine and NHR,

 where R is lower-alkyl,

with the proviso that C is not glycine or glycinamide when A is L-pyroglutamyl and B is L-histidyl.

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According to the invention, it has now been found that the peptides of the general formula I

A-B-O

in the A, B and O have the abovementioned meaning _{s are} more active and have a long-lasting activity in terms of a reduction in appetite and a reduction in food intake, as compared to the tripeptide Pyro 'glutamyl-histidyl-glycine (H (pyro ) -Glu-His-Gly-OH) isolated by Trygstad et al., Supra and Reichelt et al., Loc. Cit., Supra. The peptides of the general formula I and their pharmacologically acceptable salts are biologically equivalent to the aforementioned peptides, are therefore valuable appetitztigelnde agents for the treatment of obesity and associated pathological conditions in which a reduction in food intake is required. "These drugs also reduce gastric and pancreatic secretion, they are therefore also suitable for the treatment of pathological Conditions in which excessive gastric acid and / or pancreatic fluid formation is present. In addition, they have certain effects on the central nervous system, so that they are suitable for the acute treatment of disorders of consciousness *

The compounds according to the invention have the advantage over the natural tripeptide of being more active and having a longer lasting effect. These two advantages are of particular practical importance. Because of the lower minimum effective doses, there is a reduction in the side effects as well as the cost of producing the Compounds, Due to the long-lasting effect less frequent administration is required.

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Due to the fact that the peptides of the general formula I and their pharmacologically acceptable salts are biologically equivalent, all the preceding and following references to the peptides of the general formula I are to be understood as including the peptides themselves as well as the salts.

The invention also slit intermediates which are bonded to a solid resin support and which are used for the preparation of the compounds of general formula I »

As protecting groups in the stepwise solid phase synthesis of the intermediates, it is possible to use those groups which can be removed by one or more chemical treatments without adversely affecting the desired end product of general formula I. Preferably, these protecting groups are selected so as to form a single Can be removed together with other protective groups, for example together with the anchoring group Y defined above. A particularly suitable protective group R used in the stepwise or solid phase synthesis of the abovementioned intermediates is tert-butyloxycarbonyl (Boc).

Preferred protecting groups for protecting the imidazole nitrogen of histidine He is tosyl (Tos) or dinitrophenyl (Dnp). A suitable protecting group IP for protecting the phenylamino group in p-aminophenylalanine is the benzyloxycarbonyl group (Z). A suitable anchoring group for linking to the resin support is the group Y defined above, for example -O-CHg- • the aforementioned

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Protecting groups are stable to the reagents and under the reaction conditions used to deprotect the terminal amino group and subsequent coupling reactions. "

The term "lower-alkyl" means straight-chain or branched alkyl radicals having 1 to 3 carbon atoms «

The peptides of general formula I are prepared by stepwise solid phase synthesis starting with the terminal carboxyl group. "This method can be summarized briefly as follows. A specific amino acid chosen for C or B when C isHR with R" lower alkyl selected at the the OC- amino group has a suitable protecting group and optionally other protecting groups and is linked to the above-defined Y group with the resin support, is subjected to a deprotecting treatment on the OC- amino group and with the specific amino acid corresponding to B or A, when C has the meaning defined above KHR, coupled, being selected as the coupling agent is a suitable .Dialkyl- or Dicycloalkylcarbodiimid »The above process is repeated until the desired An * * number of amino acids is linked together» Then the protective group the terminal amino group and given If present, other protective groups and the anchoring group Y are removed. "After removal of the solvent, the desired crude peptide is obtained. The crude product is purified by chromatography, for example by gel filtration, to give the desired peptide in a pure state.

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The starting materials used for the preparation of the petides of general formula I are either commercially available compounds or can be easily prepared by methods known per se. All amino acids used for the synthesis of said peptides! are commercially available, with the exception of homo- (pyro) -glutamic acid whose preparation is described by Greenstein and Winitζ in "Chemistry of the Amino Acids", J.Wiley, New York, 196I, pp. 2407 to 2462, and β - (2-Thienyl) -alanine, the preparation of which is given in the cited reference, p. 2707, ♦

Suitable solid resin supports are chloromethylated and hydroxymethylated resins, with the former resins being preferred. With respect to the preparation of hydroxymethyl resins, see M, Bodansky and J.I. Sheehan, Chem. Ind., London, Vol. 38 (1966), p. 1597 When the chloromethylated resin is used, an ester anchoring group is formed with the amino acid O (or B if G is the above-defined UHR) with the amino acid slotted on the OL- amino group, where appropriate further protective groups are present.

(protected 0 or B) -O- CH ₂ -

A convenient method of converting the linked, protected peptide to the C-terminal (lower-alkyl) -amide is by cleavage of the protected peptide from the resin by treatment with a lower alkylamine. D. H. Coy et al., Bioehem. Biophys. Res «Common., Vol. 57 (1974)»

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Thus, the protecting groups of the formed peptide (lower alkyl) amide are removed by treatment with sodium and liquid ammonia or, preferably, by cleavage with hydrogen chloride to give the corresponding slotted peptide (lower alkyl) amide Another procedure is to carry out the cleavage by transesterification with a lower alkanol, preferably methanol or ethanol, in the presence of triethylamine to give the corresponding ester. This ester can be converted to the corresponding (lower-alkyl) amide. Subsequently, the protective groups can be removed in the manner described above. If the formation of the free carboxylic acid is desired, the cleavage is preferably carried out with hydrogen fluoride in anisole. If the formation of the acid amide is desired, the cleavage is carried out with ammonia. In this regard, reference is also made to J, M, Stewart and J, D · Young, "Solid Phase Peptide Synthesis", W, H _# Freeman & Co., San Francisco, 1969, S, 40-49.

According to one embodiment of the invention, glycine protected on the QL- amino group, preferably tert-butyloxycarbonylglycine, is coupled with a chloromethylated resin by means of a catalyst, preferably cesium hydrogencarbonate or triethylamine. After coupling of the C6 amino protected glycine to the resin support, the 06x amino protecting group is removed using, for example, trifluoroacetic acid in methylene chloride, trifluoroacetic acid alone or hydrochloric acid in dioxane. Deprotection is carried out at temperatures of about 0 ° to room temperature carried out. It can too

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other standard cleavage reagents and conditions are used to remove specific OC amino protecting groups; see. E. Schroder and K, Lubke, "The Peptides", Bd, 1, Academic Press, Hew York, 1965, S, 72-75. After removal of the O-amino protecting group, the remaining amino acids slotted on the Ct-amino group become stepwise coupled in the desired order to form the desired peptide. The protected amino acids are each introduced in a 3-fold excess in the plague phase reactor. The coupling reaction is carried out in methylene chloride or mixtures of dimethylformamide and methylene chloride. In cases where the coupling is incomplete, the coupling procedure is repeated prior to removal of the OG-amino protecting group and prior to coupling of the next amino acid to the solid phase. The successful course of the coupling reaction in the individual synthesis steps is monitored by means of the ninhydrin reaction according to E, Kaiser et al., Analyto Biochem., 34 (1970), p. 595.

After synthesis of the desired amino acid sequence, the protected peptide is removed from the resin support by treatment with a (lower alkyl) amine to form the protected peptide (lower alkyl) amide. When using dinitrophenyl or tosyl as a protecting group for the histidyl radical, these protecting groups are also removed during treatment with the (lower-alkyl) -amine. The peptide can also be separated from the resin by transesterification with a lower alkenol, preferably methanol or ethanol, in the presence of triethylamine, after which the recovered ester is purified by chromatography on silica gel. The recovered fraction may be subjected to treatment with a (lower alkyd-amine for the conversion of the lower alkyl ester, preferential

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The methyl or ethyl ester to which carboxyl-terminal (lower-alkyl) amide is subjected must be noted. It should be noted that dinitrophenyl or tosyl groups present on the histidyl radical may also be cleaved off. The remaining side chain protecting groups of the protected alkyl amide are then cleaved by the methods described above, for example by treatment with matrium in liquid ammonia or with hydrogen fluoride. The separation of the desired paptide from the resin support can also be carried out with ammonia to give the corresponding amide or with hydrogen fluoride and anisole Peptides of general formula I in which C 2 ~ aminohydroxy <! - ethyl (glycol) means are prepared by cleavage of an AB dipeptide resin with 2-aminoethanol. It should be noted that any dinitrophenyl or tosyl groups present on the histidyl radical are likewise split off in this process.

As already mentioned, the peptides of general formula I can also be obtained in the form of salts with acids, examples of which are salts with organic acids such as acetic acid, lactic acid, succinic acid, benzoic acid, salicylic acid, methanesulfonic acid and toluenesulfonic acid, polymeric acids such as tannic acid or carboxymethylcellulose , and inorganic acids, such as hydrohalic acids, for example hydrochloric acid, and sulfuric acid and phosphoric acid. Optionally, specific salts with acids can be converted into salts with other acids, for example into non-toxic, pharakologically acceptable salts, by reacting them according to R, A. Boissonnas et al., HeIv, Chim. Acta., 43 (1960), p 1349 with a suitable ionic

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Examples of corresponding ion exchange resins are cellulose-based cation exchangers, such as carboxymethyl cellulose or chemically modified, crosslinked dextran cation exchangers, for example those of the Sephadex C type, and strongly basic anion exchange resins, for example those of J ₀ P · Grstensten in and M. Winitz in Resins, "Chemistry of the Amino Acids", John Wiley and Sons, Inc., New York and London, 1996, Vol. 2, p.

The peptides of the general formula I and their salts have valuable and long-lasting effects, namely an appetite-suppressing effect, an inhibitory effect on the secretion of gastric and pancreatic juice and a CNS-activating effect. "

The appetite-reducing and anorexigenic action of the peptides of the general formula I can be determined by experiments on mice, rats or dogs, similar to the experiments described by Trygstad et al., Supra, and by Reichelt et al., Supra. The inhibitory effect of the peptides of general formula I on gastric and / or pancreatic secretion is determined on uneaten rats or preferably on unaesthetized dogs with oesophageal, gastric and pancreatic faistes or also on dogs with Heidenhain pockets} cf. B, P, Babkin, "Secretion Mechanism of Digestive Glands ", 2nd edition. Hoeber, New York, 1950, according to "Physiology", Ed. E. Seikurt, Little, Brown and Company, Boston, 1963, pp. 542 to 544s L, Olbe, Gastroenterology, Bd. 32 (1959), S 46Oi Antin et al., Journal of Comparative and Physiological Psychology, Vol. 89 (1975), p. 784i and Liebling et al., Ibid., Vol. 89 (1975), p. 955. The effect of the peptides of the general formula I to the central nervous system, in particular

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their characteristic spectrum of neurotropic effects in particular can be determined by a series of ZKS tests; see. for example A ₀ J, Kastin, R, D, Olson, AV Schally and DH Coy, Life Scineees, Vol. 25 (1975), p. 401; A _e J. Prange, G, R. Breese, J * M. Cott, BR Martin, B, R. Cooper, IC Wilson and UP Plotnikoff, Life Sciences, Bd, 14 (1974) i p 447 l and M. Brown and W. Vale, Endocrinology, Vol. 96 (1975), p 1333.

Mice, rats and dogs are preferred as experimental animals for the determination of the appetite-reducing or anorexigenic effects of drugs and for the determination of the inhibition of gastric and / or pancreatic secretion, since in many cases excellent parallels have been found between animal experiments and human clinical studies, for example, in humans under controlled oral or intragastric diet with a liquid diet} cf. HA. Jordan, Journal of Comparative and Physiological Psychology, Vol. 68 (I969), p. 498; G.A. Bray and P.L. Greenway, Clinics in Endocrinology and Metabolism, Bd * (1976), p. 455 »and H.D. Janowitz, "Role of Gastrointestinal Tract in Regulation of Food Intake," cited in "Handbook of Physiology," Section 6, Alimentary Canal., Ed. C. Code and W. Heidel, American Physiological Society, Washington, 1967.

The action of the peptides of general formula I and their pharmacologically acceptable salts as appetite suppressants and anorexigenic agents can be demonstrated using rats, rats are preferred as experimental animals for the detection of the effect of drugs that inhibit food intake. Various authors have identified the parallelism between

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See, for example, GA, Bray in "The Obese Patient," Bd, IX of "Major Problems in Internal Medicine," W, B, Saunders Company, Philadelphia, London, Toronto, 1976 In Chapter 9 entitled "Drug Therapy for the Obese Patient", under the section "Pharmacology - Effects on the Central Nervous System", on S, 364, Table 9-4, data are listed, inter alia show the reduction of feed intake in rats by a number of anorexigenic drugs. In the section "Clinical Use of Anorectic Drugs" it is shown on S, 369 in Table 9-7 that the same drugs as in Table 9-4 are also suitable for clinical use.

Due to the aforementioned properties, the peptides of the general formula I are suitable for administration to mammals in veterinary medicine and also in human medicine.

Because of their appetite-inhibiting and anorexic properties, they are useful in the treatment of obesity, including the preoperative reduction in body weight often required in large-scale obese patients. The peptides, by virtue of their properties, are also valuable in the treatment of other pathological conditions requiring a reduction in food intake and body weight, for example, in certain forms of diabetes or circulatory diseases.

Due to their inhibitory action on the secretion of gastric acid and pancreatic fluid, the paptides of general formula I are suitable for the treatment of pathalogic conditions associated with gastric and / or pancreatic hyperplasia.

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gastric acid, gastrin, pepsin or histamine, for example duodenal ulcers or acute pancreatitis.

The effect of the peptides of general formula I on the central nervous system shows a neurotrophic profile of action that differs significantly from the profiles of tricyclic antidepressants and conventional drugs. Because of their CNS-activating effect, the peptides of general formula I are valuable active ingredients for the treatment of consciousness typhus or coma due to brain injury, for example in the case of cerebral trauma or tumors, in severe cases of frostbite or in certain postoperative conditions, as well as in consciousness disorders caused by certain vascular or by excessive drug intake (for example, ₀ intoxication by drugs) are caused ·

When using the peptides of the general formula. I or their pharmacologically acceptable salts in mammals as an appetite suppressant or anorexigenic agent for the treatment of obesity or other pathological conditions requiring a reduction in dietary intake, when used as agents for inhibiting excessive gastric or pancreatic secretion or when used as CNS -activating agent, for example on mice, rats or dogs, these active substances can be used alone or together with pharmacologically compatible excipients. The ratio of drug and carrier is determined by the solubility and chemical nature of the compound, the route of administration and the usual biological practice. For example

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For example, an amount of active ingredient sufficient to reduce appetite and / or food intake may be administered orally in solid form along with carriers such as starch, sugar, certain types of clays, and the like.

Similarly, appropriate amounts can be administered orally in the form of solutions or suspensions. The compounds can also be injected parenterally. For oral or parenteral administration, sterile solutions or suspensions in a pharmacologically acceptable liquid carrier, such as water, ethanol, propylene glycol or polyethylene glycol, the other solutes or suspending agents, for example for the preparation of isotonic solutions sufficient amounts of saline or glucose, salts of bile acids , Gum arabic, gelatin, sorbitan monooleate polysorbate 80 (oleate ester of sorbitol and its ethylene oxide copolymerized anhydrides) such as Tween 80.

The dosage of the peptides of general formula I depends on the route of administration and on the particular compound chosen. Besides, it depends on the special person to be treated. In general, treatment will be started with small doses substantially below the optimal dose of the compound. Subsequently, the dosage is increased in small increments until the optimal effect is achieved under the given circumstances. In general, the compounds are administered in amounts which induce a reduction in appetite and / or food intake, an inhibition of gastric and / or pancreatic B secretion, or activation of the central nervous system without causing harmful or deleterious side effects. Such doses generally range from about 1.0 / ug to about

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250 / ug per kg of body weight per day, although, as mentioned above, these dosages may be varied. "Especially preferred are dosages in the range of from about 5 to about 100 / μg per kg per day, preferably in divided doses.

The individual pharmacological effects of the peptides of the general formula I are greater by weight than in a number of medicaments used for the same purpose. In addition, most of the clinically useful appetite suppressants have a phenethylamine-related structure and therefore have the undesirable side effects associated with this structure. The peptides of the general formula I have no Phenäthylaminstruktur and are therefore free of these side effects * They also have a low toxicity and thus ensure safe use,

When using the peptides of general formula I, preferably in the form of salts with acids, in human medicine, systemic administration, either by intravenous, subcutaneous or intramuscular injection, or sublingual or nasal administration, can be performed in conjunction with pharmacologically acceptable carriers "

Pharmaceutical preparations for oral administration can also be prepared. For this purpose, the active compounds are mixed with suitable known pharmaceutical diluents and processed in a manner known per se into preparations, such as tablets, capsules, suspensions, emulsions, solutions or dispersible powders.

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Solid preparations may be filled into capsules for oral administration. Such capsule-containing preparations may contain the solid active in admixture with solid materials which act as a buffer, for example mixed with colloidal aluminum hydroxide or calcium hydrogen phosphate, or together with an inert solid such as Lactose, containing «

Preparations in the form of tablets may be coated in a conventional manner or provided in the form of foaming or non-foaming preparations. Here, inert diluents or carriers such as magnesium carbonate or lactose may be used together with conventional disintegrants such as corn starch and alginic acid and lubricants such as magnesium stearate , be used"

For administration by nasal route in the form of drops or sprays, the peptides of the general formula I are preferably used in a sterile, aqueous carrier which also tolerates sufficient amounts of pharmacologically acceptable solutes such as buffers or preservatives, as well as for the preparation of an isotonic solution Salts or may contain glucose. The doses for intranasal administration are in the range of 1.0 to 250 / μg / kg / day or preferably from about 5 to about 100 μg / kg / day.

The peptides of general formula I can also be administered in the form of nasal powders or insufflation agents. For these purposes, the peptides in finely divided form together with a pharmacologically acceptable solid carrier, eg. B. with finely divided polyethylene glycol (Carbowax 1540), finely divided lactose or very finely divided silica (Cab-O-Sil), administered. Such agents may also other additives in finely divided

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distributed form, such as preservatives, buffers or surfactants.

The peptides of general formula I may also be administered in the form of slow release or sustained release agents as discussed below, preferably by intramuscular injection or by implantation. Such formulations are such as to release from about 5 to about 100 μg per kg of body weight per day. "

It is often desirable to administer the peptides of the general formula I continuously over long periods of time in the form of long-lasting preparations with slow active substance release or depot effect. Such forms of preparation may contain either a pharmacologically acceptable salt of the active compound having a low solubility in body fluid, for example salts with paraoic acid, tannic acid or carboxymethylcellulose, or the peptide of general formula I in the form of a water-soluble salt together with a protective agent which prevents rapid release , In the latter case, for example, the peptide of the general formula I with non-antigenic, partially hydrolyzed gelatin can be brought into the form of a viscous liquid or absorbed on a pharmacologically acceptable solid carrier, for example zinc hydroxide optionally together with protamine. The active ingredient can also be administered in the form of a suspension in a pharmacologically acceptable, liquid carrier. Further, the preparation of gels or suspensions with a non-antigenic protective hydrocolloid, e.g. B, sodium carboxymethylcellulose, polyvinylpyrrolidone,

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Sodium alginate, gelatin, polygalacturonic acids such as pectin, or certain mucopolysaccharides, together with aqueous or nonaqueous, pharmacologically acceptable liquid carriers, preservatives, or surfactants, examples of which are found in standard pharmacological manuals, such as Remington's Oharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1975 · long acting preparations by slow-release can also be obtained by microencapsulation in a pharmacologically acceptable coating material such as gelatin, polyvinyl alcohol or ethyl cellulose _o further examples of different Uberzugsmaterialien and methods of microencapsulation is to J, a. Herbig in "Encyclopedia of Chemical Technology," vol. 13, 2nd ed., Wiley, New York, 1967, pp. 436-456. Such preparations, as well as suspensions of salts of the peptides of general formula 1 which are only sparingly soluble in body fluids, are such as to release from about 5 μg to about 100 μg of the peptide per kg of body weight per day. Preferably, they are administered by intramuscular injection. Another possibility is to use some of the solid preparations listed above, for example certain sparingly water-soluble salts or dispersions or adsorbates of the salts with solid carriers, for example dispersions in a neutral hydrogel of a polymer of ethylene glycol methacrylate or similar crosslinked monomers (see US Pat 556) in the form of pellets which release the amounts of active substance indicated above. These pellets can be implanted subcutaneously or intramuscularly.

Certain peptides of the general formula I ₉ which are sparingly soluble in water can be prepared as suspensions in an aqueous or an emulsion base.

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Waaserbasis pensions are prepared with the aid of wetting agents, such as condensation products of polyethylene oxide and alkylphenols, fatty alcohols or fatty acids, and suspending agents, such as hydrophilic colloids, e.g. For example, polyvinylpyrrolidone produced. Emulsion-based suspensions are prepared by suspending the peptide with the aid of a surfactant and suspending agents in the emulsion base using the emulsifiers discussed above. Suspensions may additionally contain buffers and / or sweeteners, flavors, colors, preservatives and antioxidants.

As discussed above, pharmaceutical preparations for oral or parenteral administration are prepared from the compounds of general formula I or their pharmacologically acceptable salts so as to deliver 5 to 100 μg / kg / day, preferably in divided doses. Per dosage unit can be contained 0.3 to 15 mg of active ingredient. For parenteral administration, the use of a water-soluble peptide of the general formula I or a water-soluble salt of a sparingly water-soluble peptide of the general formula I in aqueous, sterile solution is preferred. Examples of preservatives are p-hydroxybenzoic acid methyl and propyl esters which are added together with other soluble constituents, such as sufficient amounts of sodium chloride or glucose to produce isotonic solutions. Peptides of general formula I which are sparingly soluble in water may also be administered intramuscularly in the form of solutions or suspensions in sterile, liquid, non-aqueous vehicles, for example in vegetable or animal oils. In this case, if appropriate, the abovementioned additional components of the solution or suspending agent may be contained.

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The process for preparing the peptides of the general formula I is explained in more detail below »

The carboxyl-terminal amino acid selected for G or B when C is the above-defined UHR is protected at the OC-amino group, preferably

with tert-butoxycarbonyl (R = Boc) * The protected acid is then coupled with a hydroxymethyl resin or preferably a chloromethylated resin with the aid of a catalyst, preferably cesium hydrogencarbonate or triethylamine. Subsequent to the coupling, the protecting group on the α-amino group is removed, for example using trifluoroacetic acid in methylene chloride as explained above, or by one of the other methods listed above. The removal of the protecting group is preferably carried out at temperatures of 0 ⁰ C to blank t emperat for Runaway leads ·

As the chloromethylated resin, it is preferable to use one having 1 percent crosslinking.

The carboxyl-terminated amino acid attached to the resin support by means of the anchoring group defined above, which represents the radical C or B when C is UHR, is placed in the reaction vessel of an automated peptide synthesizer programmed for the following wash cycle:

(a) methylene chloride,

(b) 33% trifluoroacetic acid in methylene chloride (2 times),

Cc) methylene chloride Cd) ethanol (e) chloroform,

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(f) 10 % triethylamine in chloroform (2 times),

(g) chloroform and (h) methylene chloride

The washed resin is then stirred in methylene chloride with a suitably protected amino acid representing the radical B or A when C is UHR. Tert-Butoxycarbonyl (Boc) is preferred as a protective group for the O-amino function. For the amino acids for the radical B, the tosyl group (Tos) is preferred as the protective group for the imidazole ^nitrogen ( ^II.sub.iDa ) of histidine. As the protective group for the phenylamino group in p-aminophenylalanine, benzyloxycarbonyl (Z) is preferable, 3-methylhistidine, β- (pyrazolyl-1) alanine and β- (2-thienyl) alanine need no protection except for the Q 1 -amino group "a substantially equimolar amount of a coupling agent such as dicyclohexylcarbodiimide or preferably diisopropylcarbodiimide is added to 'the mixture for 2 hours at room temperature and stirred (22 to 25 ⁰ C)" Then, the amino acid resin with methylene chloride (3 times) and dried. The linked amino acid protecting group is removed with 33 % trifluoroacetic acid in methylene chloride (2 times). Subsequently, steps (c) to (h) of the washing cycle explained above are carried out «

The above-mentioned steps are repeated with an appropriately protected amino acid selected for the residue A when C represents an amino acid. The protected peptide resin thus obtained is washed 3 times with methylene chloride and then 3 times with methanol and then under reduced pressure Pressure dried »One obtains practically the theoretical yield (96 per cent or above).

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For producing a peptide of the general formula I in which G is NHR, or a peptide of the general formula I, a is amino-lower alkylamide in the C, the protected peptide resin is suspended at 0 ⁰ C in the corresponding lower-alkyl amine and several Hours. Excess alkylamine is then evaporated off at room temperature. The cleaved peptide is washed out of the resin with dimethylformamide. The protected peptide is then precipitated by the addition of ethyl acetate and filtered. The protected peptide is obtained in which C is HHR or an amino acid lower alkylamide ·

For the preparation of protected peptides in which C is an amino acid amide or an amino acid lower alkyl ester, the process described above is carried out using ammonia or the corresponding alcohol + triethylamine instead of lower alkylamine. »

Removal of the protecting groups from the produced in the above manner peptide is performed by a 15- to 60-minute, preferably about 30-minute treatment of the material with hydrogen fluoride and anisole at 0 ⁰ C. The hydrogen fluoride is removed under reduced pressure and the anisole is removed by washing with ether. For the preparation of peptides of the general formula I in which G is a free amino acid, the corresponding protected peptide resin is treated in the above manner with hydrogen fluoride and anisole. At the same time the protective groups and the bond to the resin support are removed. The desired peptide of general formula I is obtained in which G is a free amino acid »

For the preparation of peptides of the general formula I ₁ in

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C 2-aminohydroxyethyl (glycol), the A-B dipeptide resin is treated with 2-aminoethanol to give directly the desired peptide of general. Formula I, in which C is glycol-ol, receives. Any dinitrophenyl or tosyl protecting groups present on the histidyl radical are removed at the same time »

The following are biological studies on the peptides of the invention »

The effect of the peptides of the general formula I on the reduction of appetite and ingestion or appetite-linging activity is determined by feeding experiments in mice and rats essentially according to the methods of Trygstad et al., Supra, and Reichelt et al., Supra »At regular intervals, the daily feed consumption is calculated to be accurate to 0.1 g and the body weight to 1 g» The other measured variables, such as oxygen consumption, body temperature, glucose and / or insulin levels in the plasma and quantitative aminograms, are determined at specific times »

The action of the peptides of the general formula I on the gastric and / or pancreatic secretion is given to rats or, preferably, to dogs which are provided with esophageal, gastric and pancreatic faistines, according to Babkin, a "0", Liebling et al. , supra or Olbe, supra, determined "a sham feeding in dogs resulted in a marked increase in acid output which was 82 percent of the maximum acid output obtained by administration of 320 ^ ug / kg / hr" histamine is reached (13.55 to 1 , 8 meq / 15 min). At the same time, there was a significant increase in serum gastrin levels from a baseline of 32 ± 5 pg / ml

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73-10 pg / ml. Sham feeding also resulted in a marked increase in pancreatic protein secretion, which was 71 percent of the maximum achievable by administration of 0.5 / μg / kg / hr, of caerulein (650 lbs. 86 mg / 15 min). The peptides of general formula I reduced the maximum acid output by about 34 percent when administered intravenously at doses of 25 to 50 μg / kg / hr 30 minutes before and during the sham flicker with no significant change in serum gastrin level and pancreas => protein excretion 38 percent «

In dogs with gastric fistulas and Heidenhain bags, gastric administration of a liver extract diet resulted in a marked increase in acid output, which was about 90 percent of the maximum achievable by administering histamine to dogs with gastric fistula, and about 35 percent of the maximum value in dogs Heidenhain · "pockets increased while serum gastrin levels significantly increased to more than 2 times the basal level from 29 lbs. 5 pg / ml to 76 ± 12 pg / ml. * When peptides of general formula I were administered by 1 -day intravenous infusion during the feeding plateau at doses of 50 / g / kg / h, the liver extract-induced acid secretion was reduced by 35% in dogs with gastric fistulas and by 41% in dogs with Heidenhain pockets, without that significant changes in serum gastrin levels occurred "The peptides of general formula I also inhibit the F SHAKE-related insulin and increases gastrin "

In dogs with pancreatic fistulas, normal feeding stimulated pancreatic HCO ^ "* to 80 percent of the maximum value obtained by administration of 3 KU / kg / hr * secretin

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achieves protein excretion to 92 percent of the maximum value, with 0.5 / ig / kg / hr. Caerulein is achievable The peptides of general formula I administered in the manner indicated above during the second hour after feeding reduced HCO 3 'output by 35 percent and protein (enzyme) output by 46 percent without nausea or other side effects »

The above results clearly show that the peptides of general formula I inhibit the brain, gastric and intestinal phases of gastric and pancreatic secretion, presumably via a suppresion of the neuro-hormonal mechanisms responsible for these secretions. The brain phase is the neural, psychic or appetite phase of gastric secretion, the inhibition of which suppresses the appetite and the anorexigenic effect of the peptides of general formula I.

Ausführungsbe ispiel

The examples illustrate the invention.

example 1

D-pyroglutamyl-L-histidyl (tosyl) -glycyl-O-CHg resin

DH- (pyro) -Glu-His (U ^lm -R ³ ) -Gly-O-OH., - Resin _< R ³ "Tos. 2.70 g (I ₉ OO mmol glycine) of the Boc-glycine resin of the formula

Boc - Gly - 0 - CH ₂ - resin

are added to the reaction vessel of an automatic peptide synthesizer (Beckman Model 990) programmed for the following wash cycle »

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Aüsführungsbeiapiele example 1

D-pyroglutamyl-L-histidyl (tosyl) -glycyl-O-CH ₂ -resin, DH- (pyro) -Glu-His (1 -R ³ ) -gly-O-CH ₂ -garboxyl, R ³ = Tos,

2.70 g (1.00 mmol glycine) of the Boc-glycine resin of the formula Boc-Gly-O-CHg resin are added to the reaction vessel of an automatic peptide synthesis apparatus (Beckmann Model 990) programmed for the following cycle :

(a) methylene chloride,

(b) 33% trifluoroacetic acid in methylene chloride (2, 1, or 25 minutes),

(c) methylene chloride,

(d) ethanol,

(e) chloroform,

(f) 10 % triethylamine in chloroform (2 times 2 minutes each),

(g) chloroform and (h) methylene chloride.

The washed resin is then stirred with 3.0 mmol of tert-butyloxycarbonyl-tosyl-histidine in methylene chloride and 3.0 mmol of diisopropylcarbodiimide are added. The mixture is stirred at room temperature for 1 hour. The peptide-resin is subjected to steps (a) to (h ) of the abovementioned washing cycle. Then, 3.0 moles of D-pyroglutamic acid are coupled in the same manner to give the title compound.

The finished tripeptide resin is washed 3 times with methylene chloride and

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subsequently washed 3 times with methanol. After drying, 2.89 g of material are obtained.

The glycine resin of this example is prepared from commercial chloromethylated resin (1% crosslink, Bio Rad Laboratories Richmond, California).

Example 2 DP.yroglutamyl-L-histidyl-glycine

Protected tripeptide resin obtained according to Example 1 is suspended in 40 ml of hydrogen fluoride and 10 ml of anisole for 45 minutes at ⁰ ° C. The hydrogen fluoride is then evaporated off with the aid of a stream of dry nitrogen. The anisole is removed by washing with ether »Moves ·

The crude peptide is purified by gel filtration on a 2.5 x 95 cm column packed with Sephadex G 15 (chemically modified, crosslinked dextran, "fine"). The appropriate fractions (320 to 340 ml) are lyophilized. 320 are obtained mg DH (pyro) -Glu-Hiaüly-ÜH in the form of a non-colored, flaky powder

The product behaves homogeneously in 4 different solvents on silica gel plates in the case of chromatic chromatography. In each case 20 to 30 / ug are applied in the form of stains. · The development is carried out with chlorine gas and anrs.chließendem spraying with starch reagent · There are the following ß ^ value of 1-butanol: acetic acid: water (4: 1: 5 upper phase ), 0.08% ethyl acetate: pyridine: acetic acid: water (5: 5: 1: 3) 0 »28j 1-butanol: acetic acid: water: ethyl acetate (1: 1: 1: 1) 0.18; 2-propanol: 1 m acetic acid (2: 1) 0.20

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The amino acid analysis gives the following value: Glu, 1.03, 1.001, His 0.99.

Example 3

L-Pyroglut am.yl-L-3'- methyl-histi-dyl-glycol-Q-CH-resin

according to the procedure described in Example 1 using 3.0 mmol t-butyloxyoarbonyl-3'-methylhistin anatieie of t-butyloxycarbonyltosylhistidine and 3 _? 0 mmol of L-pyroglutamic acid instead of D-pyroglutamic acid gives 2.90 g of the title compound.

Example ^ L-pyroglutamyl-L-O'Hnethylhistidylglycin

By the procedure described in Example 2, using 2.90 g of the title compound obtained in Example 3 and fractions aerated from batches of 280 to 320 ml, the title compound is obtained as a colorless, fluffy powder (251 mg).

The product behaves homogenously on thin silica gel plates in four solvent systems on silica gel plates. In each case 20 to 30 μg of U-form are stained. The reaction is carried out with chlorine gas and subsequent spraying with starch reagent. The following values are obtained:

1-butanol: acetic acid: water (4: 1: 5 x upper phase), υ, 04; Ethyl acetate ί pyridine: acetic acid: water (5: 5: 5: 3 ;, 0.15;

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1-uutanol: acetic acid j water: ethyl acetate (1: 1 s 1: 1)

2-Propanol: 1 M ticacetic acid (2: 1), 0,04ο The amino acid analysis gives the following value of GIu 1.05; GIy 1.00; 3-mehis, 96,

Example 5

Hach the Verrahrensweise described in Example 1 using 2.78 g Boc-D-Ala-O-CHg resin (1 mmol D-alanine), prepared from commercially available chloromethylated üarss (1 io crosslinking, Bio Rad Lab., Richmond, California), instead of ßio-oly-0-Cu ₂ resin and 3 »mmol t-butyloxy« carbonyltosylhistidine and 3.0 mmol L-pyroglutamic acid instead of D-pyroglutamic acid, obtained 3.37 g of Xitelverbindung,

Example β L-P.vrogmtamvl-JU-nisticiyl-D-alanine

Following the procedure described in Example 2 using 3 37 g of product in Example 5 and collecting the distilled mixtures of 260 to 320 ml, LH-Cpyro) -gi-i-ais-D-Ala-OH is obtained , which is further purified by chromatography on a silica gel column (1.5 x 145 cm). The development takes place with a mixture of i-butanol-acetic acid-bis-ethyl acetate (1: 1: 1: 1). The eluted extracts of 380 μl of 5 ml were collected and lyophilized. The title compound is obtained as a dry powder (39% mg).

The Proauct vernäit in the Dünnschichtchromatografie

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homogeneous in four different solvent systems on silica gel plates. In each case 20 to 30 / ug are applied in the form of stains. The development is carried out with chlorine gas and subsequent spraying with starch reagent. The following ft., - values result:

1-butanol: acetic acid: water (4: 1 ι 5 »upper phase) 0.10; Ethyl acetate: pyridine: acetic acid: water (5: 5: 1 ι 3) 0.30;

1-butanol: acetic acid; Water: ethyl acetate (1: 1: 1: 1), 0.21; 2-propanol-1m acetic acid (2: 1, 0.25 x

Example 7 D-Pyro / z: lutamyl-L ·-3- ^t- methylthiididyl-D-al-an-vinyl-CH- _R , resin

Carry out the procedure described in Example 1 using 1.39 g of Boc-D-Ala-Q-CHg resin (0.5 mmol D-alanine) prepared according to Example 5 and 1.5 mmol of t-hexane. Butyloxycarbonyl-3 ^f -methylhistidine and then 1.5 mmol of D-pyroglutamic acid, to give 1.64 g of the title compound.

Example 8 D-Byroglutamyl-L-3'-methylhistidvl-D-alanine

According to the procedure described in Example 2 using 1.64 g of the product obtained in Example 7 with 20 ml of hydrogen fluoride and 5 ml of anisole, 300 mg of the title compound are obtained as a colorless, flaky powder.

The product behaves homogeneously in the thin layer chromatography in the manner described in Example 2. It revealed

234

20.1.1983 59 950/18

the following R values;

1-Butanol: acetic acid: water (4: 1: 5) ι upper phase, 0.05} ethyl acetate: pyridine: .acetic acid: wasaer (5: 5 ί 1 t 3) »

0.15;

1-butanol: acetic acid: water: ethyl acetate (1: 1: 1: 1),

0,10j

2-propanol: 1 M acetic acid (2: 1), 0.05.

The amino acid analysis gives the following values:

Glu 1.07; AIa 1.00; 3-MeHI's 1.01.

Example 9 L-P.yroglutamyl-L-hist.vl (toeyl) -Q-CIU resin

Following the procedure described in Example 1 using 3.00 g of Boc-His (N ₂ -To) -O-CH ₂ resin (1.00 mmol histidine) prepared from commercially available chloromethylated resin (1 % cross-linking, Bio, Rad Lab. Richmond, California), instead of Boc-Gly-O-CHp resin, steps (a) to (h) of the wash cycle were performed and the washed product was treated with 3.0 mmol L-pyroglutamic acid in the presence of 3 mmol diisopropylcarbodlimide coupled. This was followed by washing the dipeptide resin with methylene chloride (three times) and with methanol (three times) and drying, giving 3 × 10 g of the peptide compound.

Example 10 L-Pyroglutamyl-L-hiatidyl-ethylamide

The product obtained according to Example 9 (3.10 g) was suspended in 20 ml of dimethylformamide at 0 ⁰ C, added 3 ml of isopropylamine and the mixture stirred at 0 ⁰ C for 18 hours. The solvent and excess ethylamine are removed under reduced pressure. The rest is mixed with 2 M of acetic acid

234263 3 - ³⁷ "

extracted and filtered to remove the resin. The peptide-containing pilate is purified by gel filtration on a 2.5 x 95 cm column packed with Sephadex G 15 (chemically modified, cross-linked dextran, "fine"). The appropriate fractions are lyophilized. 100 mg of the title compound are obtained as a colorless, fluffy powder. The product behaves when TLC homogeneous when it is examined in the manner described in Example 2, in four solvent systems · Ea, the following R _f values obtained: 1-butanol: acetic acid: water (4: 1: 5, upper phase ) 0.18; Ethyl acetate: pyridine: acetic acid in water (5: 5 ± 1: 3) 0.56;

1-butanol: acetic acid: water: ethyl acetate (1: 1: 1: 1) 0.55;

2-propanol-1m acetic acid (2: 1) 0.29. The amino acid analysis gives the following values: Glu 1.05; His 1.00; EtNH ₂ 1.03.

Example 11 L-pyroglutamyl-L-histidyl (tosyl-glycyl-O-CH-resin

Filach of the procedure described in Example 1 with replacement of D-pyroglutamic by! -Pyroglutaminsäure (3.0 mmol) is obtained 2.90 g of 'J' 'el el compound.

Example 12 L-Pyroglutamyl-L-hiBtidyl-fflycin-ethylamide

The product obtained according to Example 11 (2.90 g) is suspended in 20 ml of dimethylformamide at 0 ⁰ G, 3 nil ethylamine

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added and the mixture stirred at 0 ⁰ C for 18 hours · The solvent and excess iSthylamin is smoothly removed under reduced ^. The residue is extracted with 2 M acetic acid and filtered to remove the resin. The peptide-containing filtrate is purified according to Example 2 to give the title compound as a colorless, fluffy powder (138 mg). The product behaves homogeneously in thin-layer chromatography when analyzed in four solvent systems as described in Example 2. The following R 1 values are obtained: 1-butanol: acetic acid: water (4 * 1: 5 »upper Phase) 0.14}

Jithyl acetate: pyridine: acetic acid: water (5 * 5: 1: 3) 0.56; 1-butanol: acetic acid: water: ethyl acetate (1: 1: 1: 1)

2-propanol -1m acetic acid (2ί1) 0.25 · The analysis of the amino acid gives: Glu 1.01; GIy 1.00; His 0.97.

Example 13

Ii-pyroglutamyl-D-histidyl (tosyl) - ^ lycyl-O-QH g resin

Wake the procedure described in Example 1 using 3 »0 mmol of t-butyloxycarbonyl-tosyl-D-histidine instead of t-butyloxyoarbonyl-tosyl-histidine and of 3» 0 mmol L-pyroglutamic acid instead of ^ pyroglutamic acid to obtain 2.91 g of the title compound,

Example 14 L-PyroKlutamyl-D-histidyl-glycine

Kach the procedure described in Example 2 below

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Use of 2.91 g of the product obtained in Example 13 with 20 ml of hydrogen fluoride and 5 ml of anisole gives the title compound as a colorless, flocculated powder (125 mg).

The product behaves homogeneously after thin layer chromatography when analyzed in four solvent systems as described in Example 2. The following R values are obtained:

1-butanol: acetic acid: water (4: 1: 5, upper phase)

Ethyl acetate ί pyridine: acetic acid; Water (5: 5: 1: 3)

1-butanol: acetic acid: water: ethyl acetate (1: 1: 1: 1)

2-propanol: 1 M acetic acid (2: 1) 0.26.

The Aßiinosäureanalyse gives: Glu 1.02; GIy 1.00; His 0.97; EtNH ₂ 0.98.

Example 15

3 parts of D-pyroglutamine-L-histidyl-glycine are dissolved in 1000 parts of distilled, pyrogen-free water. To prepare an isotonic solution, a sufficient amount of sodium chloride is added. The mixture is sterilized by autoclaving and filled into ampoules. An infusion solution for therapeutic purposes is obtained.

Example 16

A mixture of 12 parts of D-pyroglutamyl-L-histidyl-glycine and 500 parts of white magnesium carbonate and 20 parts of magnesium stearate is pressed into coarse pieces. The pieces are granulated, passed through an 8-mesh sieve (2.38 mm) and pressed. The tablets obtained in this way are suitable for oral administration.

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Example 17

The mixture of 12 parts of D-pyroglutamyl-L-histidyl-glycine and 500 parts of white magnesium carbonate is granulated by admixing a solution of 2 parts of sodium dioctylsulfosuccinate in an extra amount of methanol. The granulate is (1.68 mm) passed through a 12 raeah screen and dried at 50 to 55 ⁰ G. Then the granules are again passed through a 12-mesh sieve, Hach addition of 8 parts of magnesium stearate, the mixture is compressed into tablets for oral administration.

Example 18

A mixture of 24 parts of 2-pyroglutamyl-L-histidyl-glycine, 475 parts of lactose, 94 parts of corn starch and 3 parts of magnesium stearate is pressed into coarse pieces. The pieces are crushed into a granulate which is passed through an 8-mesh sieve (2, 38 mm). The granules are then coated with a sufficient amount of a solution of 15 parts shellac and 3 parts castor oil in 800 parts ethanol. Then 3 parts of magnesium stearate are added. The granules are pressed into tablets for oral administration.

Example 19

24 parts of D-pyroglutamyl-L-histidyl-glycine are mixed in a ball mill with 576 parts of lactose. The mixture is filled into gelatine capsules. A preparation for oral administration is obtained.

Claims (5)

234263 3 m 20.1.1983 59 950/18 A method of preparing a peptide of the formula AB-C, wherein A is selected from the group consisting of L-pyroglutamyl, D-pyroglutamyl and L-homopyroglutamyl and B is selected from a group consisting of L-histidyl, D-histidyl, L- 3'-methylhistyl, L-phenylalanyl, Lp-aminophenyl-analyl and L-ß- (pyrazolyl-i) -alanyl and Q is selected from a group consisting of glycine and its lower alkyl esters, glycinamide and its lower alkylamides, D-alanine and L -β- (2'- ^1- hienyl) -alanine provided that C is not glycine or glycinamide, when-A is L-pyro-glutamyl and D is L-histidyl and optionally their pharmaceutically-acceptable salts, characterized in that the Coupling of a suitably protected, 06-amino-protected amino acid of formula C in the presence of a catalyst to a main carrier, which is selected from hydroxyethyl and chloromethylated resins, to the corresponding compound of formula (protected G) -O-GH To obtain p- (resin support), removing the 06-amino-protected group from said last-mentioned compound and coupling the resulting compound with a suitably protected, 06 amino-protected amino acid of formula B in the presence of a suitable dialkyl or Dicycloalkylcarbodiimid to the to obtain correspondingly protected peptide attached to the resin support through an anchoring group --O - CH ₂ -, removing the 06 -amino protected group from said latter compound and coupling the resulting compound with a suitably protected, oC-amino protected amino acid of formula A. in the presence of a suitable dialkyl or dicycloalkylcarbodiimide η η .. rt α ti 3 O. £ 1 234263 3 20.1.1983 59 950/18 to obtain the corresponding protected peptide bound to the resin support by means of the anchoring group -O-CHp-, remove the protecting groups from said latter compound, cleave the resulting peptide from said resin support and separate the corresponding peptide, wherein A, B and C are as defined above and optionally their reaction with an acid to the corresponding pharmaceutically acceptable salts. 2 · Method according to item 1, characterized in that the catalyst is selected from cesium hydrogencarbonate and! Triethylamine _e 3. The method according to item 1, characterized in that the cleavage of the peptide from the resin carrier by means of hydrogen fluoride in anisole and isolation of said peptide takes place as a free carboxylic acid. 4. The method according to item 1, characterized in that the cleavage of the peptide from said resin carrier by means of ammonia and isolation of said peptide takes place as acid amide. Process according to item 1, characterized in that the cleavage of the peptide from said resin support is carried out with the aid of a lower alkylamine and isolation of said peptide as the carbon-terminated (lower-alkyl) -amide. 6. The method according to item 1, characterized in that the treatment of the Boc - GIy - 0 - GH ₂ - (resin support) with 234 26 3 3 -χ- 20.1.1983 59 950/18 i'rifluoroacetic acid, followed by treatment with diethylamine, coupling the resulting compound with tert-butyloxycarbonyl-tosyl-histidine in the presence of diisopropylcarbodiimide and treating the resulting compound with trifluoroacetic acid, followed by diethylamine, coupling the resulting compound to D -Pyroglutamic acid in the presence of diisopropylcarbodiimide and separation of D-pyroglutamyl-L-hi8tidyl (tosyl) -glycyl-Q ~ CH ₂ - (resin support), treatment of said latter compound with hydrogen fluoride in anisole and isolation of D-pyroglutamyl-L- histidylglycine.