CS236662B2 - Production method of peptides - Google Patents

Description

The invention relates to a process for the production of peptides of the formula I

A - B - C (I) wherein

A is L-pyroglutamyl, D-pyroglutamyl and L-homopyroglutamyl,

B represents a residue from the group of L-histidyl, D-histidyl and L-3‘-methylhistidyl, and

C represents a residue from the group of glycine, glycinamide and its C1-6 alkylamides and D-alanine, provided that:

C has a meaning other than glycine or glycinamide if:

A is L-pyroglutamyl and

B is L-histidyl.

and their pharmacologically tolerable salts.

The invention further relates to a process for the production of compounds of the formula I, to intermediates used in the process and to the use of such compounds as appetite loss agents useful in the treatment of obesity and other pathologies in which it is advisable to reduce food intake or feed in mammals. as well as the use of these compounds in the treatment of pathologies in which excess gastric acid and pancreatic fluid excretion, such as gastric and duodenal ulcers or acute pancreatitis, and in the treatment of certain critical central nervous system conditions such as consciousness or sleepiness caused by brain injury.

The abbreviations used for amino acids and their residues as well as for protecting groups are based on the recommendations of the IUPAC-IUB Biochemical Nomenclature Commission (see Biochemistry, vol. 11 (1972), p. 1726). Examples thereof are (Pyroj-Glu = 5-oxoproline or pyriglutamic acid, homo- (pyro) -Glu = homopyroglutamic acid, His = histidine, N ⁱⁿ -3-His = 3'-methylhistidine, Phe-phenylalanine, p-NH 2 Phe = p-aminophenylalanine, N - (pyrazolyl-11Ala (N - (pyrazol-1-yl) -alanine, Gly = glycine, Gly-ol = 2-aminoethanol and Thi = β- (2-thienyl) alanine).

All amino acids have a natural or L-configuration unless otherwise indicated. D-His is a residue of D-histidine, D- (pyro) -Glu is a residue of D-pyroglutamic acid, and D-Ala is a residue of D-alanine. The abbreviations Me and Et are used for methyl and ethyl. For protecting groups, for example, the following abbreviations are used:

23666 2

Boc = target. butyloxycarbonyl,

Z == benzyloxycarbonyl,

Tos = tosyl,

Dnp - dinitrophenyl and

N ^im = histidine imidazole nitrogen;

Y is a suitable anchored on a solid resin support an attached group used in solid phase synthesis, in particular —O — CH2—

Humoral taste control, particularly by the hypothalamus, has been discussed for many years, see, for example, a review by Schally et al. in Am. J. Med. Sci., Vol. 248 (1964), page 79, and the literature cited therein Since then, the hypothalamus is known to be involved in food intake control.Electrical stimulation of the lateral part of the hypothalamus leads to hyperphagia and its destruction and inability to swallow. reducing food intake and destroying hyperfugia and obesity.

Obesity syndrome, which is induced by the administration of the gold salt of thioglucose, is in part due to hyperphagia associated with the hypothalamus damage caused by this agent. The theory of neutral control of hunger, taste and satiety is discussed by various authors. Various mechanisms are proposed:

1. Thermostatic theory

According to this theory, taste is driven by the special dynamic effect of food and its effect on body temperature.

2. Chemostatic theory

These theories assume that taste is driven by intracellular nemo extracellular concentrations of glucose (glucostatic theory), lipids (lipostatic theory) and various metabolites such as serum-amino acids.

3. Another group of theories assume that taste is regulated by the digestive tract sensations that occur when eating and in the presence of food in the stomach and intestine. These factors, which contribute to a feeling of satiety or manifest themselves by refusing to eat, include expanding the stomach by intake of non-nutritious substances. Gastric contractions may be taken into account in connection with hunger behavior, but these contractions are in animals that have been artificially damaged by the hypothalamus, having an afagic or hyperphagic state, the contractions being the same as in animals that are deprived of food and water.

However, these theories do not lead to an explanation of all the discoveries identified by the experiments. In addition, diabetic animals may feel hungry. Schally et al., In the above-cited publication, believe that the obesity occurring in the hypothalamus damage is transmitted by a hurnoral rather than a neurogenic mechanism, and that the hypothalamus may be developing a substance that participates in central taste control. Preliminary discoveries are made to point out that there is a substance present in the neurohypophysic extract that affects the dynamic phase of weight gain in mice treated with gold thioglycose.

In addition, a newer shorter review by Schally et al. on the state of research concerning the control of food intake by the hypothalamus and obesity (see Recent Progress in Hormone Research, Proceedings of the 1967 Laurentian Hormone Conference, vol. 24 (1968), p. 497, especially pages 570 and 571 and literature cited therein). These works include Schally and Mitarba in Science, Vol. 157 (1967) p. 210, which show that administration of enterogastrone isolated from the dog's duodenum will reduce food intake in mice left without food for 17 hours. Other peptides isolated from the dog's duodenum or colon, such as glucagon, secretin, glucose, and bovine serum albumin, have been shown to be ineffective. hungry stomachs can explain this, or that they were induced by the central nervous system due to the release of potalam, although positive evidence for hypertalamic neurotecutins, which are responsible for direct or indirect taste control, could not yet be made.

This positive evidence appears to have been provided by Trygstad et al. in Acta Endokrinologica, Vol. 89 (1978), p. 196. These authors have isolated a number of peptides from the urine of patients suffering from congenital general disorders of fat metabolism (hypothalamic syndrome). These peptides produced corresponding metabolic results. Nervous anorexia is associated with disorders of the hypothalamus. The primary nervous anorexia affected by the hypothalamus disrupts the hypothalamus-pituitary axis, resulting in a slight release of gonadotropin, omission of menstruation, loss of the daily rhythm of ACTH-secretion, diminished thyrotropin secretion and an initial increase and later loss of somatotropin secretion.

Urine precipitates in 25 patients with nervous appetite were chromatographed on a column filled with Sephadex G-25-gel. This was divided into four different chromatographic samples: One resembled a sample of normal controls, the other resembled schizophrenic patients, five patients with hysterical for2 3 δ 5 δ 2 my neurosis showed a third pattern and 10 'doses with a primary "type of hypothalamic *" a typical chromatogram has also been shown. Fractures affecting anorexia in mice were found only in the last group. Two appetite-influencing peptides are isolated by a plurality of chromatographic steps. When these peptides are injected into mice, one of them induces an increased appetite while the other decreases this appetite. The peptides are coated with the protein carrier of the peptide and thus protected against enzymatic degradation, allowing their isolation from the urine.

The structure of the appetite inducing peptide has been elucidated by Reichelt et al. in Neuroscience, Vol. 3 (1978), p. 1207 in close collaboration with Trygstad. It is the tripeptide H- (pyro) -Glu-His-Gly-OH, which was confirmed by synthesis.

Upon injection of 12 nmol of appetite-limiting peptide administered daily to mice for 20 days, food intake decreased from 5.7 to 3.0 g daily for about 6 months. Body weight dropped from an average of 35 g to a minimum of 24 g.

The tripeptide has no acute effect on blood glucose and serum insulin levels and appears to act on receptors in the appetite-controlling hypothalamic centers.

The appetite-promoting peptide increases the daily intake of food above 10 g, with body weight rising to 57 g on average. The structure of the appetite-supporting peptide could not be identified.

Two similar factors, which show behavior increasing and decreasing food intake, have also been isolated in patients with genetically related metabolic obesity.

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

A - B - C (I) wherein

A. B and C are as defined above, are more active and have longer sustained activity with respect to reduced appetite and food intake than is the case with the pyroglutamyl-histidyl-glycine tripeptide [H- (pyro) -Glu-His-Gly -OH], which Trygstad et al. (see above, as well as Reichelt et al. (see above isolated, as in this case.) The peptides of formula I and their pharmacologically tolerable salts, which are biologically equivalent to the above-mentioned peptides, are therefore valuable appetite-preventing agents suitable for treating Obesity and associated pathologies in which it is desirable to reduce food intake, thus reducing the secretion of gastric and pancreatic juices, and is therefore also suitable for the treatment of pathologies in which excess stomach acid and / or fluid is produced. In addition, they have some effect on the central nervous system, making them suitable for the acute treatment of consciousness disorders.

The compounds of the invention have preference over the natural tripeptide in refraction, being more active and further maintaining their effect. These, both advantages, are of particular practical importance. By virtue of the smallest " minimum effective " doses, side effects are reduced as well as the cost of producing these compounds. Due to the longer lasting effect, less frequent drug administration is required.

Because of the fact that the peptides of formula (I) and their physiologically tolerable salts are biologically equivalent, throughout the foregoing and the following reference to the compounds of formula (I) is meant to include peptides themselves as well as salts thereof.

The invention also relates to protected intermediates which are bound to a solid resin support and which are used for the preparation of the compounds of formula I.

Suitable protecting groups for the sequential solid phase synthesis of intermediates may be those which can be removed by one or more chemical treatments without prejudice to the desired end product of formula (I). Preferably, these protecting groups are selected by: they can be removed together with other protecting groups in a single step, for example together with the anchored group Y as defined above. A particularly suitable protecting group R ^2, which is used in the sequential synthesis of said intermediates in a solid phase is a tert-butyloxycarbonyl (BOCJ.

Preferred protecting groups R ³ to protect the imidazole nitrogen of histidine are tosyl (Tosj and dinitrophenyl (Dnp). A suitable protecting group R ¹ to protect the phenylamino group in p-amino-phenylalanine is benzyloxycarbonyl (Zj. The protecting groups mentioned are stable to the reagents and to the reaction conditions used to remove the amino-terminal protecting group and the subsequent coupling reactions.

The term "lower alkyl" means a straight or branched alkyl radical having 1 to 3 carbon atoms.

The peptides of formula (I) are produced by sequential solid phase synthesis starting from the terminal carboxyl group. This procedure can be briefly expressed as follows:

The process according to the invention consists in binding a protected amino acid based on the C-protected residues on the α-amino group in the presence of a catalyst supported from a group of hydroxyethylated and chloromethylated resins to give the corresponding compound of formula (protected C residue). - O - CH 2 - (resin support).

the α-amino protecting group is removed from this compound, and the resulting compound is bound to the protected amino acid based on the residues with the meanings indicated for B, protected on the α-amino group in the presence of C 2 -C 10 dialkylcarbodiimide or dicycloalkylcarbodiimide. From 6 to 10 carbon atoms in cycloalkyl groups, to form the corresponding protected peptide attached to the resin support anchored with an -O-CH2-group, the α-amino protecting group is removed from this compound and the resulting compound binds with a protected amino acid on the base of the residues ## STR6 ## in the presence of a C2 -C10 dialkylcarbodiimide or a C6 -C10 dicycloalkylcarbodiimide, to form the corresponding protected peptide attached to the resin support in the O-CH 2 group, the protecting groups are removed from the compound, the resulting peptide of formula (I) is cleaved from the resin support and the corresponding peptide is isolated, optionally treated with a pharmaceutically acceptable acid to give the corresponding pharmaceutically acceptable salt of the peptides.

A more detailed explanation of the method according to the invention is given below in a broader context.

An amino acid specially selected for C or B wherein C is NHR ¹ with R ¹ in the meaning of lower alkyl having an appropriate protecting group or optionally other protecting group on the α-amino group and bonded to a resin support as defined above by Y is subjected to and to further bind with a certain amino acid for B or A when C is as defined above for NHR ¹ , wherein a suitable dialkylcarbodiimide or dicycloalkylcarbodiimide is selected as the coupling agent. This procedure is repeated until the required number of amino acids has been linked to each other. The amino-terminal protecting group as well as any other protecting groups present and the anchored group Y are then removed. Removal of the solvent yields the desired crude peptide. The crude product is purified by chromatography, for example by gel filtration, to give the desired peptide in a pure state.

The starting materials used to produce the peptides of formula (I) are either commercially available compounds or can be readily prepared according to methods known per se. All amino acids used to synthesize said peptides are commercially available, with the exception of homo- (pyro-gltaminic acid), the preparation of which has been described by Gréenstein and Winitz in "Chemistry of the Amino Acids", J. Wiley, New York, 1961, p. 2462 and (3- (2-thienyl) alanine, the preparation of which is mentioned in the aforementioned literature reference on page 2707).

Suitable solid resin carriers are chloromethylated and hydroxymethylated resins, with chloromethylated resins being preferred. With respect to the production of hydroxymethyl resins, reference is made to the article by M. Bodansky and JT Sheehan, Chem. Ind., London, Vol. 38 (1966), p. 1597. Chloromethylated resins are commercially available. When using a chloromethylated resin, an ester-anchored group with amino acid C (or B when C is the above-defined NHR ¹ group) protected on the α-amino group is formed, optionally with additional protecting groups present.

(protected by C or Bj - O - CH2 -

An effective method of converting bound protected peptides to the C-terminal (lower alkyl amide) is by cleaving the protected peptide from the resin by treatment with a lower alkylamine, see DH Coy et al., Biochem. Biophys. Res. Commun., 57 (1974), p. Finally, the protecting group of the resulting peptide (lower alkyl) amide is desulfurized by treatment with sodium and liquid ammonia, or preferably by cleavage with hydrogen chloride, to yield the corresponding peptide. Another method of production is that the cleavage is carried out by transesterification with a lower alkanol, preferably methanol or ethanol, in the presence of triethylamine to give the corresponding ester, which ester can be converted to the corresponding (lower alkyl) amide, and finally Remove protective groups as prescribed above and a free carboxyl group, the cleavage is preferably carried out with hydrogen fluoride in anisole, and if the formation of the acid amide is desired, the cleavage is carried out with ammonia. In this regard, reference is made to the publication by J. M. Stewart and J. D. Young, "Solid Phase Peptide Synthesis", W. H. Freeman CO., San Francisco, 1969, pp. 40-49.

According to one embodiment of the invention, the α-amino-protected glycine, preferably tert-butyloxycarbonylglycine, is bound to the chloromethylated resin by means of a catalyst, preferably ces236BB2 bicarbonate or triethylamine. After binding of the α-amino-protected glycine to the resin support, the α-amino protecting group is removed, for example using trifluoroacetic acid in methylene chloride, trifluoroacetic acid alone or hydrochloric acid in dioxane. The deprotection is carried out at a temperature of about 0 ° C to room temperature. Other standard cleavage reagents and standard conditions can also be used to remove specific α-amino protecting groups (see E. Schroder and K. Lubke, "The Peptides", Vol. 1, Academic Press, New York, 1965, p. 72). to 75). Upon removal of the .alpha.-amino protecting group, the remaining amino acids protected on the .alpha.-amino group gradually bind in the desired order to form the desired peptide. The desired amino acids are always introduced in triple excess into the solid phase reactor. The coupling reaction is carried out in methylene chloride or in a mixture of dimethylformamide and methylene chloride. In the event that the binding is not complete, the binding procedure is repeated before the α-amino protecting group is removed and the next amino acid is bound to the solid phase. The success of the coupling reaction in the individual synthesis steps is monitored by the ninhydrin reaction of E. Kaiser et al., Analyt. Biochem. (1970), p. 595.

After synthesis of the desired portion of the amino acid, the protected peptide is removed from the resin support by treatment with a lower alkylamine to form the protected peptide- (lower alkyl) amine. When dinitrophenyl or tosyl is used as a protecting group for a histidyl residue, these protecting groups are also removed during treatment with a lower alkylamine. The peptide can also be separated from the resin by transesterification with a lower alkanol, preferably methanol or ethanol. in the presence of triethylamine, and then the ester obtained by chromatography on silica gel. The desired fraction may be subjected to treatment with a lower alkylamine to convert the lower alkyl ester, preferably the methyl ester or ethyl ester, into the carboxyl terminal lower alkyl amide. It should be noted that the dinitrophenyl or tosyl groups present on the histidyl residue are optionally cleaved. Thus, the remaining side chain protecting groups of the protected alkylamine are cleaved according to the above-described process, for example by treatment with sodium in liquid ammonia or hydrogen fluoride. The separation of the desired peptide from the resin support can also be carried out with ammonia to form the corresponding amide or with hydrogen fluoride and anisole to give the corresponding free acid. The peptide of formula I wherein C is 2-aminohydroxyethyl (Gly-ol) is produced by cleavage of the A-B-dipeptide resin with 2-aminoethanol. It should be noted that in this process, the dinitro10 phenyl or tosyl groups present on the histidyl residue are optionally cleaved.

As already mentioned, the peptides of the formula I can also be obtained in the form of acid salts. Examples thereof are salts with organic acids such as acetic acid, lactic acid, succinic acid, benzoic acid, sulfuric acid, methanesulfonic acid and toluenesulfonic acid, polymeric acids such as tannin, carboxymethylcellulose and inorganic acids such as hydrohalic acids such as hydrochloric acid as well as such as sulfuric acid and phosphoric acid.

Individual acid salts may optionally be converted to other acid salts, for example, those which are nontoxic and pharmacologically tolerable when, according to R. A. Boissonnas et al., Helv. Chim. Acta., Vol. 43 (1960 J, p. 1349) performs treatment with a suitable ion exchange resin.

Examples of suitable ion exchange resins are cellulose cation exchangers such as carboxymethylcellulose or chemically modified, cross-linked dextran cation exchangers, such as the Sephadex C type, as well as the strongly basic anion exchange resins reported by JP Greenstein and M. Winitz in "Chemistry of the Amino Acids", John Wiley & Sons. Inc., New York and London, 1961, Vol. 2, page 1456.

The peptides of the formula I and their salts have valuable and long-lasting effects, namely an anorexia-inducing effect, an effect preventing the secretion of gastric and pancreatic juices, as well as an active action on the central nervous system.

The appetite-limiting effect and anorexigenic effect of the peptides of formula (I) can be determined in an attempt to eat in mice, rats or dogs by the experiment described by Trygstad et al. and von Reichel spol. The suppressive effect of peptides of the formula I on the secretion of juices in the stomach and pancreas is determined on waking rats or preferably on pharyngeal, gastric and pancreatic dogs, or also on dogs with a Heidsnhain sac, see Β. P. Babkin, "Secretion Mechanism of Digestive Glands'", 2nd ed., Β. P. Hoeber, New York, 1950, citing "Physiology", Hrsg. E. Selkurt, Little, Brown and Company, Boston, 1963, pp. 542-544: L. Olbe, Gastroenterology, Vol. 32 (1959J, p. 460; Antin et al., Journal of Comparative and Physiological Psychology, vol. 89 (1975), p. 784; and Liebling et al., Ibid., Vol. 89 (1975), p. 955.

The effect of the peptides of formula I on the central nervous system, particularly their characteristic spectrum of nerve effects, can be determined by a number of central nervous system tests, see, for example, A.J. Kastin, R. D. Olson, A. V. Schally and D. H. Coy,

It

Life Scineces, Vol. 25 (1975), p. 401; A. J. Prange, G. R. Breesé, J. M. Cott, B. R. Marin, B. R. Cooper, I. C. Wilson and N. P. Plotniikbff, Life Sciences, Vol. 14 (1974), p. 447 and M. Brown and W. Vale, Endocrinology, vol., 96 (1975), p. 1333.

Mice, rats and dogs are preferred as test animals to determine appetite-reducing and anorexigenic effects of drugs, as well as to inhibit the secretion of gastric and / or pancreatic juices, since in many cases there are excellent parallels between test animals and clinical examinations in humans, for example in humans receiving controlled oral or intragastric fluid diet, see HA Jordan, Journal of Comparative and Physiological Psychology, Vol. 68 (1969) 498; G.A. Bray and F.L. Greenway, Clinics In Endocrinology and Metabolism, Vol. 5 (1976), p. 455 and H. D. Janowltz, "The Role of Gastrointestinal Tract in Regulation of Food Intake", cited in "Handbook of Physiology", paragraph 6, Alimentary Canal., Hrsg. Code R. and W. Heidel, American Physiological Society, Washington, 1967.

The effect of the peptides of formula I and their pharmacologically tolerable salts as appetite suppressants and anorexigenic agents can be demonstrated using rats. Rats as test animals are preferred to demonstrate the effect of drugs that limit food intake.

Various authors have established parallelism between rat and human results, see, for example, G. A. Bray in The Obese Patient, Vol. IX of "Major Problems in Internal Medicine", W. B. Saunders Company, Philadelphia, London, Toronto, 1976.

Chapter 9 of the title “Drug Therapy for the Obese Patient” provides data in the paragraph “Pharmacology - Effects on the Central Nervous System” on page 364, Table 9–4, indicating a reduction in food intake in rats induced by a number of anorexigenic drugs. . In the paragraph "Clinical Use of Anorectic Drugs" on page 369, Table 9-7, it is stated that the same drugs as in Table 9-4 are also suitable for clinical use.

Because of these properties, the peptides of formula I are suitable for administration to mammals in veterinary as well as human medicine.

Peptides based on appetite-reducing and anorexigenic properties are useful in treating obesity including preoperative weight loss, which is often useful in obese patients before major interventions. Peptides are also valuable because of their properties for the treatment of other pathological conditions that require a reduction in food intake and body weight, for example, in certain forms of diabetes or circulatory diseases.

The peptides of the formula I are suitable because of their effect of limiting the secretion of gastric acid and pancreatic fluids *

abdominal to treat pathological conditions that come with gastric and / or pancreatic hypersecretion of gastric acid or gastrin, pepsin or histamine, for example in gastric ulcers or acute pancretitis.

The effect of the peptides of formula I on the central nervous system shows a neurotrophic effect profile that is significantly different from that of tricyclic antidepressants and conventional drugs. By virtue of their central nervous system activity, peptides of formula I are valuable active ingredients for the treatment of consciousness disorders or long-lasting, artificially uninterrupted profound unconsciousness due to brain injury, for example, cerebral trauma or cancer, severe frostbite or certain postoperative as well as in a consciousness disorder caused by certain vascular diseases or by excessive drug use (for example, drug intoxication).

When using peptides of formula (I) or pharmacologically tolerable salts thereof in mammals as an appetite suppressant or anorexigenic agent for treating obesity or other pathological conditions requiring reduced food intake, when used as a means to prevent excessive secretion of gastric or pancreatic juices or when used as active agents for the central nervous system, for example in mice, rats or dogs, the active compounds may be used alone or together with pharmacologically compatible carriers.

The drug / carrier ratio is determined by the solubility and chemical nature of the compound, the route of administration, and usual biological practice. For example, an amount of active ingredient that is sufficient to reduce appetite and / or food intake may be administered orally in solid form together with carriers such as starches, sugar, selected types of clays and the like.

In a similar manner, an appropriate amount may be administered orally in the form of a solution or suspension. The compounds may also be administered by parenteral injection. For oral or parenteral administration, sterile solutions or suspensions in pharmacologically compatible liquid carriers, such as water, ethanol, propylene glycol or polyethylene glycol, are used which contain liquid or suspending agents, for example, to produce isotonic solutions of a sufficient amount of table salt or glucose, bile salts, gum arabic, gelatin, sorbitan oleate, polysorbate 80 (sorbitol oleate and its anhydride copolymerized with ethylene oxide), such as Tween 80.

The dosage of the peptides of the formula I depends on the route of administration and the specially selected compound and, moreover, depends on the individual person to be treated. Generally, treatment is initiated with small dosages which are substantially below the optimum dose of the compound. Thereafter, the dosage is increased in small increments until an optimum effect under the conditions is obtained.

Generally, it is treated with the compound in an amount that causes a reduction in appetite and / or food intake, prevents secretion of the stomach and / or pancreas, or central nervous system activation without causing harmful or disadvantageous side effects. Such dosages generally range from about 1.0 to 250 µg per kilogram of body weight per day, although such dosages may vary. Particularly preferred is a dosage in the range of about 5 to about 100 μξ per kilogram per day, preferably in divided doses.

The individual pharmacological effects of the peptides of formula I are greater in weight than in a number of drugs that are used for the same purpose. In addition, the clinically most commonly used appetite suppressants have a phenathylanine-related structure and therefore have undesirable side effects associated with this structure. The peptides of formula I do not have a phenethylamine structure and therefore do not have side effects thereof. In addition, they are slightly toxic and guarantee certain uses.

When using peptides of the formula I, preferably in the form of acid salts, in human medicine, systemic administration can be performed, either by intravenous, subcutaneous or intramuscular injection. or by sublingual or nasal administration, together with pharmacologically tolerable carriers.

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

Solid formulations may be filled into capsules for oral administration. Such capsule-filling compositions may contain a solid in admixture with solid materials having a buffer effect, for example in admixture with colloidal aluminum hydroxide or dicalcium phosphate, or may be together with an inert solid such as lactose.

Tablet formulations may also be coated by conventional means or may be formulated as foaming or non-foaming formulations. In this case, inert diluents and / or carriers, such as magnesium carbonate or lactose, can be used together with conventional disintegrants, such as corn starch and alginic acid and a binder, such as magnesium stearate.

For nasal administration in the form of drops or sprays, the peptides of formula I are used, preferably in a sterile aqueous carrier, which may also contain other solutions.

solid substances such as buffers or preservatives, as well as sufficient amounts of physiologically tolerable piglets or glucose for the preparation of isotonic solutions. Dosages for intranasal treatment are in the range of about 1.0 to 250 µg / kg / d 'or preferably about 5 to about 100 (Ug / kg / d).

The peptides of formula I may also be administered in the form of nasal powders or inhalation agents. For this purpose, the peptides in finely divided form are administered together with pharmacologically compatible solid carriers, for example, comminuted polyethylene glycol (Carbowax 1540), finely divided lactose, or very fine silica (Cab-O-Silj). particles, such as preservatives, buffers or surfactants.

The peptides of formula (I) may also be administered in the form of long-acting, slow release or depot active agents, as further explained, preferably by intramuscular injection or implantation. Such forms of administration are designed to release about 5 to about 100 g of active ingredient per kilogram of body weight per day.

It is often desirable to administer the peptides of the formula I continuously or over a prolonged period of time in the form of long-acting, slow-release or depot-active preparations. Such formulations may contain either a pharmacologically tolerable salt of the active compound with poor solubility in body fluids, for example pamoic acid, tannin or carboxymethylcellulose salts, or a water-soluble salt salt peptide of formula I, together with a high release preservative.

In the latter case, for example, the peptide of formula (I) may be converted into a viscous liquid with the non-antigenic partially hydrolyzed gelatin, or the peptide of formula (I) may be absorbed on a pharmacologically compatible solid carrier such as zinc hydroxide together with iprotamine.

The active ingredient may also be in the form of a suspension with a pharmacologically compatible liquid carrier. It is also possible to prepare gels or suspensions with a non-antigenic protective hydrocolloid, for example sodium carboxymethylcellulose, polyvinylpyrrolidone, sodium alginate, gelatin, polygalacturonic acids, such as pectin, or certain mucopolysaccharides, together with aqueous or non-aqueous, active ingredients, pharmacologically compatible or active substances. Examples of such formulations are provided in conventional pharmacological guides such as Remington's Pharmaceutical 236666 cal Sciences, Mack Publishing Co., Easton, Pa., 1975.

Long-acting slow release formulations are also obtained by microencapsulation in a pharmacologically compatible coating material such as gelatin, polyvinyl alcohol or ethylcellulose. Further examples of coating materials and the microencapsulation method are provided by J. A. Herbig in "Encyclopedia of Chemical Technology", Vol. 13, 2nd ed., Wiley, New York, 1967, pages 436-456.

Such preparations, as well as suspensions of the salts of the peptides of formula I which are poorly soluble in body fluids, are produced by releasing about 5 to about 100 µg of peptide per kilogram of body weight per day. Another possibility is to produce some new formulations, for example those sparingly water-soluble salts or dispersions or adsorbates of salts with solid carriers, for example dispersions in a neutral hydrogel of an ethylene glycol methacrylate polymer or similar crosslinked monomers (see U.S. Pat. No. 3 55,1556), in the form of pellets which release the aforementioned amount of the active ingredient, which pellets can be implanted suhcutaneously or intramuscularly.

Certain peptides of formula I that are poorly water soluble may be prepared as a suspension in water or in an emulsion base. Aqueous-based suspensions are prepared with wetting agents, such as the condensation products of polyethylene oxide and alkylphenols, aliphatic alcohols or fatty acids, or suspending agents, such as hydrophilic colloids, for example polyvinylpyrrolidone.

Emulsion-based suspensions are prepared by suspending the peptide with a wetting agent and a suspending agent in the emulsion base using the above emulsifying agents. The suspensions may additionally contain buffer and / or sweeteners, flavoring agents, coloring agents, preservatives and antioxidants.

As explained above, medicaments for oral or parenteral administration are prepared from the compounds of Formula I or their pharmacologically tolerable salts so that 5 to 100 µg per kilogram / d can be used, preferably in divided doses. The dosage unit may contain between 0.3 and 15 μ% of the active substance. For parenteral administration, water-soluble peptides of the formula I or water-soluble salts of a peptide of the formula I which are sparingly soluble in water, in aqueous sterile solution, are preferably used.

Examples of the preservative are p-hydroxybenzoic acid methyl ester and propyl ester, which, together with other soluble constituents such as for the preparation of isotonic solutions, are added in a sufficient amount, based on sodium chloride or glucose. Peptides of formula (I) which are poorly soluble in water can 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 doing so, they may optionally contain the abovementioned additional solution components or suspending agents.

The method of production of the peptides of the formula I is explained in more detail below.

The carboxy-terminal amino acid selected for C or B when C is NHR ¹ as defined above is protected on the α-amino group, preferably with tert-butoxycarbonyl (R ² = BOCJ). The protected acid is then bound with a hydroxymethyl resin or The chloro-methylated resin is preferably hydrogenated with a catalyst, preferably cesium bicarbonate or triethylamine, and then deprotected at the .alpha.-amino group using, for example, trifluoroacetic acid in methylene chloride, as described above, or by any other method described above. it is preferably performed at a temperature of 0 ° C to room temperature.

Commercially available products are preferably used as chloromethylated resins (1% crosslinking).

A carboxylic acid-terminated amino acid linked to a resin support by means of an anchor group as defined above, which is C or B, if C is NHR ¹ , is placed in a reaction vessel in an automatic peptide synthesis device programmed for the following wash cycle:

(a) methylene chloride, (b) 33% trifluoroacetic acid in methylene chloride (2 times), (c) methylene chloride, (d) ethanol, (e) chloroform, (f) 10% triethylamine in chloroform (2k, calc), (gj chloroform) and (h) methylene chloride.

Washed resin was then stirred in methylene chloride with the appropriately protected amino acid represented by the radical A or B, if C represents a radical NHR ^first As a protecting group for α-aminofunction, tert-butylcarbonyl (BOCJ) is preferred. For amino acids for residue B, the tosyl group (Tos) as the protecting group is preferred for imidazole nitrogen (N ™) of histidine.

As the protecting group for the phenylamino group in p-aminophenylalanine, benzyloxycarbonyl (Z) is preferred; 3-Methylhistidine, (S-pyrazolyl-1-alanine and β- (2-thienyl) alanine need no protection except for the α-amino group, essentially an equivalent amount of binding agent such as dicyclohexylcarbodiimide or preferably diisopropylcarbodiimide is added and the mixture is mixed. After stirring for 2 hours at room temperature (22-25 ° C), the 3X-linked amino resin is washed with methylene chloride and the protecting group is removed from the bound amino acid by treatment twice with 33% trifluoroacetic acid in methylene chloride, followed by steps (cj to (h)). washing cycle explained above.

The above steps are repeated with the corresponding protected amino acid selected for residue A when C is an amino acid. The prepared peptide resin protected in this way is washed three times with methylene chloride and then three times with methanol and then dried under reduced pressure. The product is obtained practically in theoretical yield (96% or more).

To produce a peptide of formula I wherein C is NHR ¹ , or a peptide of formula I wherein C is a lower alkyl amino acid, the protected peptide resin is suspended at 0 ° C in the corresponding lower alkylamine and stirred for several hours. The excess alkylamine is then evaporated at room temperature. The cleaved peptide is washed with dimethylformamide from the resin. The protected peptide is then precipitated by the addition of ethyl acetate and then filtered off. A protected peptide is obtained wherein C is NHR ¹ or a lower alkyl amino acid amino acid.

To produce protected peptides in which C is an amino acid amide or a lower alkyl amino acid ester, the above-described procedure is performed using ammonia or the corresponding alcohol and triethylamine instead of the lower alkylamine.

The deprotection of the peptide produced by the process is carried out by treating the material with hydrogen fluoride in amisole at 0 ° C for 15 to 60 minutes, preferably about 30 minutes. The hydrogen fluoride was removed under reduced pressure and the anisole was washed with ether. To produce peptides of formula I wherein C is a free amino acid, the corresponding protected resin-bound peptide is treated as described above with hydrogen fluoride and anisole. The protective groups and the bond to the resin support are simultaneously removed. This yields the desired peptide of formula I wherein C is a free amino acid.

To produce peptides of formula I wherein C is 2-amino-hydroxyethyl (Gly-ol), the resin-bound Aβ-dipeptide is treated with 2-aminoethanol to directly obtain the desired peptide of formula I wherein C is Gly-ol . The dinitrophenyl or tosyl protecting groups optionally present on the histidyl residue are simultaneously removed.

The following biological experiments are again shown on the peptides of the invention.

The effect of the peptide of formula (I) on the reduction of appetite and food intake and / or the appetite-enhancing effect is determined by feeding experiments in mice and rats essentially following the procedures described by Trygstad et al. and Reichelt et al. and which are mentioned above. Daily food intake to 0.1 g and body weight to 1 g are determined at regular intervals. Other measurement data, such as oxygen consumption, body temperature, plasma glucose and / or insulin levels, and the aminogram are examined at a specific point in time. .

The effect of peptides of formula I on the secretion of juices by the stomach and / or pancreas is determined in rats (or preferably in dogs having pharyngeal, gastric or pancreatic fever according to Babkin (cited above), Liebling et al. the above-mentioned publication] or Olbe (see above) Feeding in dogs leads to a marked increase in acidity, which is 82% of the maximum acidity value that can be achieved by administering 320 µg / kg / h histamine (13.55 At the same time, there is a significant increase in serum gastrin levels from baseline of 32 ± 5 pg / ml to 73 ± 10 pg / ml. The feeding impression also leads to a marked increase in protein secretion by the pancreas, which is 71% of the maximum value and is achievable by administering 0.5 µg / kg / h caerulein (650 ± 86 mg / 15 min).

Peptides of the formula I reduce the maximum acidity by about 34% without a significant change in serum gastrin and pancreatic secretion by about 38% by intravenous infusion at a dose of 25 to 50 µg / kg / h 30 minutes before and during feeding.

In dogs with a gastric fistula and a Heidenhain sac, gastric administration of feed from the liver extract leads to a significant increase in acidity, which is about 90% of the maximum that can be achieved with histamine to dogs with a gastric fistula and about 35% of a maximum in dogs with a Heidenhain sac whereas serum gastrin levels increase significantly to more than twice the baseline, from 29 ± 5 pg / ml to 76 ± 12 pg / ml.

When administering a peptide of formula I one-hour IV infusion during the intake of food at a dose of 50 .mu.g / kg / hr to decrease acid secretion induced by food having a liver extract in dogs provided with gastric fistulae 35 a / _o and in dogs with Heidenhain pouch by 41% without there was a significant change in serum gastrin levels. The peptides of formula (I) also prevent an increase in the level of insulin and gastrin caused by food.

In dogs with pancreatic fistula, normal food stimulates pancreatic HCO3 production to 80% of the maximum value achievable by 3KU / kg / h secretin and protein secretion to 92% of the maximum hr2 3 6 G 6 2. , 5 µg per kilogram / h caerulein. The peptides of formula I administered in the above manner reduce the production of HCO ^- 3 by 35% and protein (enzyme) production by 46% during the second hour after feeding, without inducing vomiting or other side effects.

From the above, it is apparent that the peptides of formula I prevent the brain, stomach and intestinal phases from secreting the stomach and pancreas juices by suppressing the neurohormonal mechanisms responsible for this secretion. The brain phase is the neutral, psychic or taste phase of gastric secretion, the suppression of which indicates appetite suppression and the anorexigenic effect of the peptides of formula I.

The examples illustrate the invention.

Example 1

A method for producing D-pyroglutamyl-L-histidyl- (j tosyl-glycyl-O-CH resin [DH (about pyr) -Glu-His (N ^{im -R3)} -Gly-resin CHT-R ³ = Tos]

2.70 g (1.00 mmol) of glycine Boc-glycine-resin of general formula

The Boc-Gly-O-CH2-resin is introduced into a reaction vessel forming an automatic peptide synthesis device that is programmed for the following wash cycle:

(a) methylene chloride, (b) 33% trifluoroacetic acid in methylene chloride (2 times, 1 or 25 minutes), (c) methylene chloride, (d) ethanol, (e) chloroform, (f) 10% triethylamine in chloroform (2 times 2 times each) minutes), (g) chloroform and (hj methylene chloride).

The washed resin was then stirred with 3.0 mmol of tert-butyloxycarbonyltosylhistidine in methylene chloride and 3.0 mmol of diisopropylcarbodiimide were added. The mixture was stirred at room temperature for 1 hour. The resin-bound peptide is subjected to the wash cycle described above in steps (a) to (hj.) Then 3.0 mmol of D-pyroglutamic acid is weighed in the same way to give the title compound.

The finished resin bound tripeptide was washed three times with methylene chloride and then three times with methanol. After drying it gets

2.89 g of material.

The resin-bound glycine of this example is made from a commercially available chloromethylated resin (1% crosslinking i.

• 20

Example 2

Process for the preparation of D-py, roglutamyl-L-histidylglycine

2.89 g of the resin-bound tripeptide obtained in Example 1 were suspended in 40 ml of hydrogen fluoride and 10 ml of anisole for 45 minutes at 0 ° C. The hydrogen fluoride was then evaporated using a stream of dry nitrogen. The anisole was removed by washing with ether.

The crude peptide was purified by gel filtration on a 2.5 X 95 cm column packed with Sephadex G 15 (fine chemically modified, cross-linked dextran). The corresponding fraction (320-340 mL) was lyophilized to yield 320 mg DH- (pyro) -Glu -His-Gly-OH in the form of a colorless flaky powder.

The product behaves as homogeneous by thin layer chromatography in 4 different solvents on silica gel plates. It is always present in the form of spots at 20 to 30 µg.

Development is carried out with chlorine gas and then sprayed with starch reagent. Reach these values R _f -r- 1-butanol: acetic acid: water (4: 1: 5, upper phase), 0.08; ethyl acetate: pyridine: acetic acid: water (5: 5: 1: 3) 0.28; 1-butanol: acetic acid: water: ethyl acetate (1: 1: 1: 1) 0.18; 2-β, petroleum alcohol: 1 m acetic acid (2: 1) 0.20.

Amino acid analysis achieves the following values:

Glu 1.03, Gly 1.00, His 0.99.

Example 3 parts of D-pyroglutamyl-L-histidyl-glycine are dissolved in 1000 parts of distilled, pyrogen-free water. Sufficient sodium chloride is added to produce the isotonic solution. The mixture is sterilized in an autoclave and filled into ampoules. An infusion solution for therapeutic purposes is obtained.

He attaches

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

Example 5

A mixture consisting of 12 parts of D-pyroglutamyl-L-histidyl-glycine and 500 parts of white magnesium carbonate is granulated by mixing with a solution of 2 parts of sodium dioctylsulfosuccinate in a sufficient amount of methanol. The Granu2 3.8 S 6 2 is passed through a 12 mesh screen and dried at a temperature of up to 55 [deg.] C. The granulate is then passed through a 12 mesh screen once more. 8 parts of magnesium stearate are compressed into tablets for oral administration.

Example 6

The mixture consisting of 24 parts of D-pyroglutamyl-L-histidyl-glycine, 475 parts of lactose, 94 parts of corn starch and 3 parts of magnesium stearate is compressed into coarse pieces. The pieces are ground to a granulate which is passed through an 8 mesh screen. The granules are then coated with a sufficient amount of a solution consisting of 15 parts of shellac and 3 parts of castor oil in 800 parts of ethanol, followed by 3 parts of magnesium. stearic acid salts The granulate is compressed into tablets for oral administration.

Example 7 parts of D-pyroglutamyl-L-histidyl-glycine are mixed in a ball mill with 576 parts of lactose. The mixture is filled into gelatin capsules. A preparation for oral administration is obtained.

Example

L-pyroglutamyl-L-3‘-methylhistidyl-glycyl-O-CH2-resin

In the same manner as described in Example 1, except that 3.0 mmol of tert-butyloxycarbonyl-3‘-methylhistidine was used instead of tert-butyloxycarbonyltosylhistidine and 3.0 mmol of L-pyroglutamic acid instead of D-pyroglutamic acid were used. Thus, 2.90 g of the title compound are obtained.

Example 9

Process for preparing L-pyroglutamyl-L-3 L-methylhistidyl-glycine

In the same manner as described in Example 2, using 2.90 g of the product prepared as described in Example 8 and collecting the fractions eluting from 280 to 320 mL, 251 mg of the title compound was obtained, which was obtained as a colorless flaky powder.

This product is homogeneous by staining as determined by thin layer chromatography, using a 20 to 30 [mu] g charge in 4 solvent systems on silica gel plates. Developing is effected with chlorine gas and then sprayed with starch reagent.

The following R _£ values are obtained: 1-butanol: acetic acid: water (4: 1: 5, upper phase) 0.04, ethyl acetate: pyridine: acetic acid: water (5: 5: 1: 3) 0 15,1-butanol: acetic acid: water: ethyl acetate (1: 1: 1: 1) 0.10, 2-butanol: 1 M acetic acid (2: 1) 0.04.

Amino acid analysis gave the following values: Glu 1.05, Gly 1.00, 3-MeHis, 0.96.

He did

L-pyroglutamyl-L-histidyl (tosyl) -D-alanyl-O-CH2-resin

In the same manner as described in Example 1, except that 2.78 g of Boc-D-Ala-O-CH2-resin (1. mmol of D-alanine) made from commercially available chloromethylated resin (1% crosslinking instead of Boc) was used. Gly-O-CH2-resin, 3 mmol of tert-butyloxycarbonyltosylhistidine and 3.0 mmol of L-pyraglutamic acid instead of D-pyroglutamic acid gave 3.37 g of the title compound.

They did

Process for preparing L-pyroglutamyl-L-histidyl-D-alanine

In the same manner as described in Example 2 except that 3.37 g of the product obtained as described in Example 10 was used and collected fractions eluting from 260 to 320 ml, LH- (pyro) Glu-L-His- D-Ala-OH, which was further purified by silica gel column chromatography (1.5 X 145 cm), developing 1: 1: 1: 1 mixtures of 1-butanol, acetic acid, water, and ethyl acetate. fractions eluting from 380 to 560 ml, which were lyophilized to give 390 ml of the title compound as a colorless powder.

This product is homogeneous by staining as determined by thin layer chromatography, using a 20 to 30 [mu] g charge in 4 solvent systems on silica gel plates. Developing is effected with chlorine gas and then sprayed with starch reagent. Reach these values R _f: 1-butanol: acetic acid: water (4: 1: 5, upper phase) 0.10, ethyl acetate: pyridine: acetic acid: water (5: 5: 1: 3) 0 , 30, 1-butanol: acetic acid: water: ethyl acetate (1: 1: 1: 1) 0.21, 2-butanol: 1 M acetic acid (2: 1) 0.25.

Amino acid analysis gave the following values: Glu 1.04, Ala 1.00, His 0.95.

Example 12

Process for the preparation of D-pyroglutainyl-L-3‘-methylhistidyl-D-alanyl-O-CH2-resin

In the same manner as described in Example 1, except that 1.39 g was used

Boc-D-Ala-O-CH2-resin (0.5 mmol of D-alanine) produced as described in Example 1036862, using 10 mmol of tert-butyloxycarbonyl-3'-methylhistidine and then 1.5 mmol D-pyroglutanoic acid to give 1.64 g of the title product.

Example 13

Process for the preparation of D-pyroglutamyl-L-3‘-methylhistidyl-D-alanine.

In the same manner as described in Example 2, except that 1.64 g of the product prepared as described in Example 12 was used, with 20 ml of hydrogen fluoride and 5 ml of anisole, 300 mg of the title compound was obtained as a colorless flake powder.

The product is homogeneous according to thin layer chromatography carried out by the same procedure as described in Example 2. They reach these values R _f: 1-butanol:

: acetic acid: water (4: 1: 5, upper phase) 0.05, ethyl acetate: pyridine: acetic acid: water (5: 5: 1: 3) 0.15, 1-butanol: acetic acid: water : ethyl acetate (1: 1: 1: 1) 0.10, 2-butanol: 1M acetic acid (2: 1) 0.05.

Amino acid analysis gave the following values: Glu 1.07, Ala 1.00, 3-MeHis 1.01.

Example 14

Process for preparing L-pyroglutamyl-L-histldyl (tosyl) -O-CH2-resin

In the same manner as described in Example 1 except that the treatment was subjected to 3.00 g of Boc-His (N ^im -Tos) -O-CH2-resin (1.00 mmol of histidine) made from commercially available chloromethylated resin (1). % cross-linking) instead of Boc-Gly-O-CH2-resin, using the wash cycle of steps (a) to (h) and further washing the washed product with 3.0 mmol of L-pyroglutamic acid in the presence of 3.0 mmol of diisopropylcarbodimide followed by washing the dipeptide resin three times with methylene chloride and washing three times with methanol and drying to give 3.10 g of the title product.

Example 15

Process for preparing L-pyroglutamyl-L-histidylethylamide

3.10 g of the product obtained as described in Example 14 are suspended in 20 ml of dimethylformamide at 0 ° C, 3 ml of ethylamine are added and the mixture is stirred at 0 ° C for 18 hours. The solvent and excess ethylamine were evaporated under reduced pressure and the residue was extracted with 2 M acetic acid and filtered to remove the resin. The peptide-containing filtrate was purified by gel filtration on a 2.5 X 95 cm column of fine, chemically modified cross-linked dextran (Sephadex G 15) and the appropriate fractions were lyophilized to give 100 mg of the title compound as a colorless flaky powder.

This product is homogeneous by thin layer chromatography, determined by the same procedure as described in Example 2, using a four-solvent system. Reach these values R _f: 1-butanol: acetic acid: water (4: 1: 5, upper phase) 0.18, ethyl acetate: pyridine:

: acetic acid: water (5: 5: 1: 3) 0.56, 1-butanol: acetic acid: water: ethyl acetate (1: 1: 1: 1) 0.55, 2-butanol: 1M acetic acid (2: 1) 0.29.

Amino acid analysis gave the following values: Glu 1.05, His 1.00, EtNHz 1.03. Example 16

L-pyroglutamyl-L-histidyl (tosyl) -glycyl-O-CH2-resin

In the same manner as described in Example 1, except that D-pyroglutamic acid was replaced with 3.0 mmol of L-pyroglutamic acid, 2.90 g of the title product was obtained.

Example 17

Process for preparing L-pyroglutamyl-D-histidyl (tosyl) -glycyl-O-CH2-resin

In the same manner as described in Example 1, except that tert-butyloxycarbonyltosylhistidine (3.0 mmol) and D-pyroglutamic acid (D-pyroglutamic acid 3.0 mmol) and L-pyroglutamic acid (3.0 mmol) 3.0 g of the title product are obtained. in the title.

Example 18

Process for preparing L-pyroglutamyl-D-histidyl-glycine

In the same manner as described in Example 2, except that 2.91 g of the product obtained as described in Example 17 was used, with 20 ml of hydrogen fluoride and 5 ml of anisole, 125 mg of the title compound was obtained as a colorless flaky powder.

The product is homogeneous according to thin layer chromatography, as determined by the same procedure as described in Example 2. They reach these values R _f: 1-butanol: acetic acid: water (4: 1: 5, upper phase) 0.14 ethyl acetate: pyridine:: acetic acid: water (5: 5: 1: 3) 0.56, 1-butanol: acetic acid: water: ethyl acetate (1: 1: 1: 1) 0.51, 2 butanol: 1M acetic acid (2: 1) 0.25.

Amino acid analysis gave the following values: Glu 1.01, Gly 1.00, His 0.97.

23G5S2

Example 19

Process for the preparation of L-pyrogrutamyl-L-histidyl-glycinethylamide

2.90 g of the product obtained as described in Example 18 are suspended in 20 ml of dimethylformamide at 0 ° C, 3 ml of ethylamine are added and the mixture is stirred at 0 ° C for 18 hours. The solvent and excess ethylamine were evaporated under reduced pressure and the residue was extracted with 2 M acetic acid and filtered to remove the resin. The peptide-containing filtrate was purified as described in Example 2. 138 mg of the title compound was obtained as a colorless flaky powder.

This product is homogeneous by thin layer chromatography, determined by the same procedure as described in Example 2, using a four-solvent system. Reach these values R _f: 1-butanol: acetic acid: water (4: 1: 5, upper phase] 0.15, ethyl acetate: pyridine: acetic acid: water (5: 5: 1: 3) 0 54, 1-butanol: acetic acid: water: ethyl acetate (1: 1: 1: 1) 0.53, 2-butanol: 1M acetic acid (2: 1) 0.26.

Amino acid analysis gave the following values: Glu 1.02, Gly 1.00, His 0.97, EtNHz 0.98.

Claims (6)

A process for the production of peptides of the general formula I, A — B — C (I) wherein A is L-pyroglutamyl, D-pyroglutamyl and L-homopyroglutamyl, B represents a residue from the group of L-histidyl, D-histidyl and L-3‘-methylhistidyl, and C represents a residue from the group of glycine, glycinamide and its C1-6 alkylamides and D-alanine, provided that: C has a meaning other than glycine or glycinamide if: A is L-pyroglutamyl and B is L-histidyl, as well as pharmaceutically acceptable salts thereof, characterized in that the protected amino acid based on the residues designated for C, protected on the? -Amino group, in the presence of a catalyst supports a hydroxyethylated and chloromethylated resin support resins to form the corresponding compound of formula (protected C residue) -O-CH 2 - (resin support), removing the amin-amino protecting group from this compound and binding the resulting compound to a protected amino acid based on the residues defined for B, α-amino-protected, in the presence of a (C 2 -C 10) dialkylcarbodiimide or (C 6 -C 10) dicycloalkylcarbodiimide, to form the corresponding protected peptide attached to the resin support anchored with an "O-CH 2" moiety thereof is removed The? -amino protecting group and the resulting compound bind with the protected? -amino acid based on the residues as defined for A, protected at the? -amino group in the presence of C 2 -C 10 dialkylcarbodiimide or C 6 -C 10 dicycloalkylcarbodiimide in cycloalkyl groups to form the corresponding protected peptides attached to the resin support anchored with an —O — CH2— group, deprotecting the compound, resulting peptide of formula I cleaved from the resin support and isolating the corresponding peptide, optionally treated with a pharmaceutical with an acceptable acid and the resulting pharmaceutically acceptable salt of the peptides is isolated. 2. A process according to claim 1 wherein the catalyst is cesium bicarbonate and triethylamine. 3. The method of claim 1 wherein the peptide is cleaved from the resin support by treatment with hydrogen fluoride in anisole and the peptide is isolated in the form of a free carboxylic acid. 4. The method of claim 1 wherein the peptide is cleaved from the resin support by treatment with ammonia and the peptide is isolated as an acid amide. 5. The method of claim 1, wherein the peptide is cleaved from the resin support by treatment with a C1 -C6 alkylamine and the peptide is isolated in the form of C1 -C6 C-terminal alkyl amides. 6. The process of claim 1 for the preparation of D-pyroglutamyl-L-histidyl-glycine, wherein the compound of formula 2 3 N 8 8 2 The Boc-Gly-O-CH2- (resin support) was treated with hydrofluoric acid and then with triethylamine, the resulting compound was bound with tert-butyloxycarbonyltosylhistidine in the presence of diisopropylcarhodiimide, and the resulting compound was treated with hydrofluoric acid and then with triethylane. D-pyroglu tamyl-L-histidyl (tosyl) -glycyl-O-CH2- (resin support) was isolated and treated with fluoro hydrogen in anisole and isolated.