DD289545A5 - PROCESS FOR THE PRODUCTION OF NEW PEPTIDES - Google Patents

Abstract

The invention relates to a process for the preparation of novel peptides of the general formula (I) and salts thereof: HTyrR2AspAlaIlePheThrAsnSerTyr {peptides preparation; Secretion enhancement growth hormone; Drug}

Description

and optionally of pharmaceutical preparations having GRF activity or optionally of compositions which can be used in animal breeding, characterized in that the correspondingly protected amino acids or amino acid fragments are coupled in appropriate order by methods known in peptide chemistry, the protective groups are split off and, if desired, the resulting peptide is salted transferred to a salt obtained or the peptide is released or converted into another salt and optionally the compounds of general formula I or a pharmaceutically acceptable salt or metal complex thereof with customary inert pharmaceutical carriers and / or excipients and brings to a galenic form or optionally Compounds of the general formula I or a salt or metal complex thereof which can be used in animal breeding are mixed with suitable excipients which are customary in animal breeding and brin to a form which can be used in animal breeding gt.

2. The method according to claim 1 for the preparation of compounds of general formula (I) and salts thereof, in which R ¹³ VaI, R ¹⁵ GIy or! eu and R ^{28 are} Ser and R ² , R ¹¹ , R ²⁷ and Y have the meaning given in claim 1, characterized in that one uses the corresponding starting materials.

3. The method of claim 1 for the preparation of compounds of general formula (I) and salts thereof, in which R ² Ala, R ¹¹ Arg, R ¹³ VaI, R ¹⁵ Gly, R ²⁷ Met or Never and R ²⁸ Asn and

Y has the meaning given in claim 1, characterized in that one uses the corresponding starting materials.

4. The method of claim 1 for the preparation of compounds of general formula (I) and salts thereof, in which R ² Ala, R ¹¹ Arg, R ¹³ He, R ^{1S is} Gly, R ^{27 is} Met or Never and R ^{28 is} Asn and

Y has the meaning given in claim 1, characterized in that one uses the corresponding starting materials.

5. The method of claim 1 for the preparation of compounds of general formula (I) and salts thereof, in which R ² Ala, R ¹¹ Arg, R ¹³ VaI, R ¹⁵ Gly, R ²⁷ Nie and R ²⁸ Ser and Y are the im Claim 1 specified meaning, characterized in that one uses the corresponding starting materials.

For this 1 page drawing

Field of application of the invention

The invention relates to a process for the preparation of novel peptides which are used as medicaments in human medicine and veterinary medicine.

Characteristic of the known state of the art

The new peptides which can be prepared according to the invention are analogous to the factor promoting growth hormone secretion. The secretion of growth hormone-promoting hormones (growth hormone releasing factor, also referred to as "GRF") and analogues thereof play a very important role in both medicine and veterinary medicine as well as in animal breeding

Compounds are used in human medicine to treat abnormal conditions associated with the deficiency or decreased secretion of growth hormone, and to promote growth, enhance feed utilization, improve meat-to-fat ratio, and increase milk production in veterinary and animal breeding used.

Shortly after isolation of the naturally occurring "natural" human GRF (also referred to as "hGRF"), attempts have been made to clarify the correlation between chemical structure and biological activity 44-membered peptide) of the amino acid sequence of GRF isolated from the organism of several mammals (eg, pig, cattle, sheep) .The active center of the molecules was generally found to be that containing the amino acids in positions 3-21 The biological activity of the shorter peptide chains is generally inferior to the efficacy of the natural hormone, however, the experiments have shown that even the hGRF peptide (1-29hNH ₂ (ie, the natural peptide amide of human origin containing the 1-29th amino acids of the N-terminal calculated) has a favorable activity compared to natural hGRF (1-44) -NH ₂ [Nature 300, 286 (1982); Med. Chem. 30,219 (1987); Peptides, page 481 (Editor: D. Theodoropoulos, Publisher: Walter de Gruyter, Berlin, New York, 1988); Proceedings of the Ninth Am. Peptide Symposium (Publisher: Pierce Chem. Co., Rockford, Illinois, 1985) page 707; Biochem. Biophys. Res. Commun. 123,854 (1984)). The experiments carried out with analogs of hGRF- (1-29) -NHj have demonstrated that, viewed from a biological activity point of view, the N-terminal part is more important than the C-terminal part and the activity of the peptides in general lower than the activity of the peptide amides [J. Med. Chem. 28, 181 (1985). It has further been found that the biological activity of peptides consisting of fewer members than the natural hormone can be favorably influenced if some amino acids of the peptide chain are replaced by other amino acids. Accordingly, z. B. by the exchange D-Ala ² and Leu ^{16 increases} the activity or biological half-life of the analogues [J. Med. Chem. 28, 181 (1985)]. After further experience, replacing the N-terminal near or located in positions 1-11 amino acids against the D-antipodes to increase the biological effect. It is further evident in the above publications and in the experimental results of D. Coi and coworkers [Peptides 7, Suppl. 1.49-52 (1986)] that the 27th amino acid of the naturally occurring hormone (ie, the readily oxidizable Methionine) can be replaced by the structurally closest amino acid (ie, norleucine) without breaking the activity.

Following a general experience relevant to practical application, those GRF analogs containing at least 29 amino acids are effective. According to US Pat. Nos. 4,518,586 and 4,528,190, the above processes can be observed not only in human hormones but also in several hormones of animal origin. It is further known from the above publications that the 1-29. Amino acid sequence of the naturally occurring hormone in the human and porcine hormones is identical. The 1-29. The amino acid sequence of the hormone isolated from bovine and goat differs from the above hormones in that in position 28, instead of serine, the amino acid asparagine is present. The sequence of 1-29. Amino acids in sheep GRF are similar to those of bovine or goat derived hormone, but this amino acid sequence contains the amino acid isoleucine at position 13 instead of valine. According to the technical literature [J. Dairy Be. 70, 2511 (1986) and 71, 92 (1988)], by administering human GRF (1-44) -NH2 and GRF (1-29) -NH ₂ , despite the different structure, both the quality of bovine milk production are also affected quantitatively favorable.

Object of the invention

The invention provides methods of producing novel peptides analogous to the factor promoting secretion of growth hormone and having a significantly greater activity compared to naturally occurring hormones.

Explanation of the essence of the invention

The present invention is based on the object, methods for the production of GRF analogues, which are at least as effective or even more effective than the naturally occurring hormone or its, from the amino acids 1-29.

existing sequence.

The present invention is based on the finding that the thirty amino acids analogues containing at the 30-position instead of glutamine the amino acid γ-Aminomilchsäureamid and their 1-29. Amino acid sequence corresponds to the naturally occurring sequence or contains partially known and partially new amino acid substitutions, the efficiency of naturally occurring GRF or GRF (1-29) -NH2 or GRF (1-29) -OH significantly exceed.

The three-letter abbreviations of the amino acids used in the present specification correspond to the recommendations of IUPA IV -IVB Biochemical Nomenclature [J. Biol. Chem. 247,977 (1972)]. Unless we say otherwise, the amino acids have an L configuration. The other abbreviations used have the following meanings:

Gaba residue of γ-amino lactic acid

Boc tert. butoxycarbonyl

BHA benzhydrylamino group

TFA trifluoroacetic acid

DIC diisopropylcarbodiimide

HOBt 1-hydroxy-benzotriazole

HF hydrogen fluoride

MPLC medium pressure liquid chromatography

HPLC liquid chromatography with high performance

TLC thin layer chromatography

hGRF natural pituitary isolated GRF (44-membered peptide)

hpGRF-44 from human τη pancreatic isoPorter GRF, its amino acid composition with that of GRF

Pituitary origin is consistent with bGRF derived from pituitary-derived natural GRF (44-membered peptide), whose 1-29. Amino acid sequence with

same as that of goat-isolated hormone oGRF from sheep pituitary isolated natural GRF (44-membered peptide)

GH growth hormone

STH release secretion (excretion) of the growth hormone, the subject of the invention is a process for the preparation of new peptides of the general formula (I) and salts thereof

"H-Tyr-R'-Asp-Ala-Ile-Phe-Thr-Asn-Sdr-Tyr

-R "-Lys-R ¹³ -Leu-R ^1B -Gln-Leu-Ser-Ala-Arg- (I)

-Lys-Leu-Leu-Gln-Asp-Me-R "-R ²⁸ -Arg-Gaba-Y _}

R ^{2 is} Ala or D-Ala,

R "is Arg or D-Arg,

R ' ³ VaI or He means

R ^{15 is} Gly, Leu, Phe or a valence bond,

R ²⁷ Met or Never means

R ²⁸ Ser, D-Seroder Asnist, and

Y is OH or NH ₂ ,

according to which the correspondingly protected amino acids or amino acid fragments are coupled in a suitable order according to methods known in peptide chemistry, the protective groups are split off and, if desired, the resulting peptide is converted into a salt or the peptide is released from a salt obtained or converted into another salt.

According to an advantageous embodiment of the process according to the invention, compounds of the general formula (I) are prepared in which R ^{13 is} VaI, R ^{16 is} Gly, Leu and R ^{28 are} Ser and R ² , R ", R" and Y have the above meaning.

These compounds are analogous to human and porcine hormones.

According to another advantageous embodiment of the process according to the invention compounds of general formula (I) are prepared in which R ² Ala, R "Arg, R ' ³ VaI, R ¹⁵ Gly, R ²⁷ Met or Nie and R ^{28 are} Asn and Y is the These compounds are analogous to the hormones derived from bovine and goat.

According to a further advantageous embodiment of the process according to the invention, compounds of the general formula (I) are prepared in which R ^{2 is} AlA, R "is Arg, R ¹³ lie, R ^{16 is} Gly, R ^{27 is} Met or Nie and R ^{28 is} Asn and Y is the above These compounds are analogous to the hormone isolated from sheep.

In another embodiment of the process according to the invention, compounds of the general formula (I) are prepared in which R ^{2 is} Ala, R "is Arg, R ^{13 is} VaI, R ^{16 is} Gly, R ^{27 is} Nie and R ^{28 is} Ser and Y is the above meaning Has.

The above compounds of the general formula (I) have particularly advantageous properties.

The novel compounds of the general formula (I) can be prepared according to the invention by the methods known in peptide chemistry. These compounds may be prepared in liquid phase by condensation of the appropriately protected amino acids, or, more preferably, of the fragments formed from the corresponding protected amino acids, in the appropriate order. However, the compounds of general formula (I) can be prepared according to a particularly advantageous embodiment of the process according to the invention with a solid-phase synthesis. In this case, first the correspondingly protected Gaba is attached to the resin, after which the correspondingly protected amino acids or fragments formed therefrom are coupled in a suitable order.

The novel compounds prepared according to the invention can be used in human medicine, veterinary medicine and animal breeding to promote the secretion of growth hormone. For this purpose, the compounds of general formula (I) or salts thereof are completed in the form of pharmaceutical preparations or compositions useful in veterinary or animal breeding.

The invention further relates to a process for the preparation of pharmaceutical preparations with GRf action (with the growth hormone secretion promoting effect), after which a compound of general formula (I), wherein R ² , R ", R ' ³ , R ¹⁵ , R ²⁷ , R ²⁸ and Y have the abovementioned meaning, or a pharmaceutically acceptable salt or metal complex thereof mixed with customary inert pharmaceutical carriers and / or excipients and brings to a galenic form.

The invention furthermore relates to a process for the preparation of compositions which can be used in animal breeding, according to which a compound of the general formula (I) in which R ² , R ", R ¹³ , R ¹⁶ , R", R ²⁸ and Y are as above or a salt or Metallkorbplex used in animal breeding thereof mixed with suitable additives customary in animal breeding and brings to a form applicable in animal breeding.

The pharmaceutical preparations according to the invention or compositions which can be used in veterinary or animal breeding can also contain the compounds of general formula (I) in the form of salts or metal complexes.

Suitable salts are acid addition salts (for example hydrochlorides, hydrobromides, sulfates, phosphates, maleates, acetates or citrates etc.). The complexes of the compounds of general formula (I) may, for. 8. Complexes formed with zinc or iron.

The pharmaceutical preparations and compositions useful in animal breeding may be e.g. B. administered intravenously, intramuscularly, subcutaneously, intranasally or orally.

The orally administered preparations may, for. B. be completed in the form of tablets. These preparations can in addition to the active ingredient adjuvants, eg. As carriers (such as calcium carbonate), binders (such as tragacanth, corn starch or gelatin), explosives (such as alginic acid) and lubricants (such as magnesium stearate) contained. Liquid preparations can also change the taste

containing improving agents. The usable in animal breeding, orally administrable compositions may, for. B.

Be feed premixo.

The injectable preparations may contain, in addition to the active ingredient for injection purposes, suitable liquid carriers (eg, an isotonic saline or phosphate buffer solution).

The active ingredient may also be completed in the form of sustained release preparations. To prepare these preparations, the active ingredient is embedded in known polymers which ensure the delayed action.

The daily dose of the active compounds according to the invention depends on several factors (eg species of the individuals to be treated, mode of administration, the effect to be achieved, etc.). The daily dose is generally between 1 and 100 mg / kg of body weight. However, this interval is merely informative and the drug dose actually used may be both lower and higher.

The biological activity of the compounds of general formula (I) is demonstrated by means of in vitro and in vivo test methods.

In vitro experiments

The excretion (secretion) of GH is measured as a function of the concentration of the test compound. The results thus obtained are compared with those obtained with the administration of naturally occurring hpGRF or hGRF (1-29) -NH ₂ , respectively.

The amount of secreted GRF is determined according to radioimmunanalysis (reliability limit 95%).

Under in vitro conditions, the novel compounds of general formula (I) produced according to the invention exhibit approximately fifty times the activity determined by cell culture and approximately twice as high as the known naturally occurring hpGRF-44 after superfusion experiments.

The results of the experiments carried out on cell cultures are illustrated in FIG. The experiments are carried out with a s.g. Monolayer cell culture of rat pituitary made. The diagram summarizes the results of 18 trials. The activity of hpGRF-44 is compared with the activity of the compound of the invention Nle ^ .Gaba ^ hGRFd-SOhNHj (designated in the Figure with the code number SZB-4-179). GH secretion is illustrated as a function of the concentration of the administered peptides. The "basal level" corresponds to the natural GH secretion of the cells It can be seen from the figure that in order to achieve the same secretion of growth hormone (ng / culture), the known hpGRF-44 is administered at a concentration approximately fifty times higher than the compound prepared according to the invention must be.

In vivo experiments

Sexually mature Wistar R Amsterdam rats (body weight 250-30Og) are given a 60 mg / kg i. p. Narcotized dose of pentobarbital. The active compounds according to the invention are dissolved in a 0.9% by weight aqueous sodium chloride solution and injected intravenously (i.v.), intramuscularly (i.m.) or subcutaneously (s.c.) 20 minutes after the anesthesia. The jugular vein of the animals is taken before (-1 minute) and after (5, 15 and 30 minutes) the administration of the drug blood samples. The amount of secreted growth hormone (GF) is determined by radioimmunoanalysis (reliability limit 95%).

The test compound used is Nle ²⁷ , GaLia ³⁰ -hGRF (1-30) -NH 2. The reference compound used is the hGRF (1-29) -NH ₂ . The test compound and the standard (reference compound) are administered iv at a dose of 2 mg / kg body weight. Upon administration of both compounds, an increase in secretion was observed. However, the compound produced according to the invention exerts a much greater effect than the known reference compound. The GH level of the rats treated with the compound according to the invention is 164% after 5 minutes, 155% after 10 minutes and 204% after 30 minutes (100% = GH blood levels of the animals treated with the reference compound). The tests also use the following test compounds:

Compound 1: D-Ala ^ Nle ^ Gaba ^ -hGRFd-SOJ-NHj

Compound 2: D-AlaM-eu ^ Nle ^ Gaba ^ -hGRFd-SO-NHj

Compound 3: D-Ala ² , D-Arg ¹¹ , Leu ¹⁶ , Nle ", Gaba ³⁰ -hGRF (1-30) -NH ₂

The reference compound (standard) is the hGRF (1-29) -NH2.

The results obtained are shown in Tables I-III. In the tables, the percentage increase in activity is also given.

Intravenously administered hGRR1-29) -NH ₂ enhances secretion of growth hormone. The percentage increase in activity of the intravenously administered compounds of general formula (I) is compared with the values obtained using hGRF (1-29r-NH ₂₎ .

When administered intramuscularly and subcutaneously, the hGRF (1-29) -NH2 was virtually ineffective in the doses used. Therefore, in these test experiments, the activity of the compounds of general formula (I) was characterized by the percentage increase in the amount of urinary growth hormone present in the blood (basal GH value).

Table I

Plasma GH-Splegel (ng / ml ± SE) after intravenous administration

Time In a dose of 0.8 pg / kg verobrei Verbin increase uhtes peptide increase Peptide administered at a dose of 2 mg / kg Verbin increase Verbin raised stabili minutes default Training 1 % Verbin % default Training 1 % 2 hung% 71.64 2 124.29 100.12 -1 80.46 ± 18.42 96.90 125.22 ± 14.28 ± 11.38 ± 8.96 η = 11 ± 18.43 ± 13.12 n = 7 η = 10 n = 13 535.54 172 » n = 9 151 » n = 9 1,801.43 170 » 1,427.90 135 » +5 311.00 ± 103.86 468.92 1,061.11 ± 213.84 + 170.45 ± 34.32 η = 11 ± 68.86 ± 165.60 .- = 7 η = 10 n = 15 240.73 257 » n = 9 217 » n = 9 1 ', 77,0O 299 » 614.38 156 » + 15 93.60 ± 63.65 203.51 393.13 ± 137.81 ± 99.30 ± 7.54 η = 11 ± 38.10 ± 64.62 n = 7 η = 10 n = 15 74.27 140 η = 9 140 n = 9 293.00 268 » 133.20 120 +30 48.73 ± 9.20 71.49 109.22 ± 28.63 ± 14.56 ± 3.37 n = 11 ± 7.62 ± 10.39 n = 7 n = 7 n = 15 n = 9 n = 9

η: number of treated animals

Table II

Plasma GH-Splegel (ng / ml ± SE) after intramuscular administration

Time "A" Connection 1 "B" % "A" Connection 2 "B" % "A" Connection 3 ..B " % minutes 49.60 %. 48,80 62.85 % 51.73 51.80 % 43,50 -1 ± 6.07 ± 5.38 ± 14.67 ± 6.07 ± 1.46 ± 12.43 n = 9 n = 8 n = 9 η = 8 η = 5 n = 4 119.40 242.20 493 122.09 658.86 1290 108.50 247.00 568 +5 ± 24.83 238 ± 51.34 ± 20.22 193 ± 56.50 ± 16.42 209 ± 27.76 n = 5 n = 5 n = 9 η = 8 n = 6 n = 4 120.00 351.00 716 138.04 409.17 786 98.67 213.75 489 + 15 ± 14.38 238 ± 58.1? ± 26.44 219 ± 89.49 ± 18.53 189 ± 45.77 56,00 149.40 304 70.19 184.02 353 54,50 110.66 254 +30 ± 17.30 110 ± 63.03 ± 9.13 110 ± 32.93 ± 2.63 105 ± 28.56 η = 5 η = 5 η = 9 η = 8 η = 5 η = 4

"A": administered at a dose of 2 mg / kg .B ": administered at a dose of 5pg / kg%: percentage increase compared to baseline (GH level measured in -I.minute) n: number of animals tested

Table III Plasma GH level (ng / ml ± SE) after subcutaneous administration

Time . "A" Connection 1 % "Β" % ,, A " Connection 2 % "B" % Connection 3 "A" % minutes 68.17 89.67 68.57 63.30 63.33 -1 ± 9.61 ± 17.63 ± 8.33 ± 7.20 ± 16.70 η = 6 η = 9 n = 7 n = 11 n = 3 285.00 419 410.67 455 304.00 443 555.85 880 237.50 374 +5 ± 68.15 ± 55.22 ± 45.49 ± 106.57 ± 33.40 n = 5 n = 9 n = 7 n = 11 n = 6 376.67 552 429.22 476 203.29 294 438.92 695 193.17 305 +15 ± 78.63 ± 69.48 ± 48.89 ± 98.15 +42.35 η = 6 n = 9 n = 7 n = 11 n = 6 145.67 213 137.78 152 69.14 100 140.38 222 83.83 132 +30 ± 34.45 ± 30.36 ± 9.16 ± 29.85 ± 16.87 η = 6 η = 9 n = 7 n = 11 n = 6

.A ": administered at a dose of 5pg / kg .B": administered at a dose of 12.5pg / kg%: percentage increase compared to the basal value (GH value measured in the -1st minute) n: number of experimental animals

The advantages of the novel compounds which can be prepared according to the invention can be summarized as follows:

- much greater activity compared to naturally occurring hormones;

- Gaba occurs in the living organism (eg in the nervous system), is not alien to the organism, its metabolism is cleared up;

as a result of the spatial filling, Gaba is linked to excellent spacer in the solid-phase peptide syntheses and as the first amino acid to the polymer carrying the peptide, thus ensuring good synthesis efficiency;

- The replacement of methionine in the 27-position to Never is chemically advantageous for the synthesis.

embodiments

Further details of the method according to the invention can be found in the following examples, without limiting the scope of protection to these examples.

example 1

Preparation of Nle ^<27 , Gaba ³⁰ -hGKr ('l-3O) -NH ₂

The peptide is prepared using a Beckman 990B peptide synthesizer on a benzhydrylamine resin (capacity: 0.65 meq / g). The protected Boc-Gaba-OH amino acid is used in an excess of 3 equivalents, calculated on the capacity of the resin, using the DIC condensing agent and the HOBt catalyst in an amount equivalent to the protected amino acid. The reaction time of the attachment of the protected Boc-Gaba-OH is 12 hours. Thereafter, the resin-protected amino acid compound is checked with a ninhydrin reaction to check the completeness of attachment to the resin. The attachment of the protected Boc-Gaba-OH amino acid to the resin is generally complete already after the first coupling. However, if the ninhydrin reaction is positive (indicating that the amino groups of the benzhydrylamine resin are not fully substituted) attachment to the resin can be completed by the symmetric anhydride method. (Due to the increase in weight, the capacity of the resin is 75-80% of the value given by the manufacturer.) The Boc-Gaba-BHA resin is split and neutralized in the usual way. Thereafter, the peptide synthesis is carried out stepwise according to the following scheme:

1. Wash with CH ₂ Cl ₂ three times for 2 minutes

2nd cleavage with 33% TFA / CH ₂ Ci ₂ once 2 minutes

3. einmal25Minuten Spaltungmit33% TFA / CH ₂ CI ₂

4. Wash with CH ₂ Cl ₂ three times for 2 minutes

5. Wash with ethanol three times for 2 minutes

6. Wash with chloroform 3 times 2 minutes

7. Neutralize with 10% Et ₃ N / CHCl _{3, 2} times 3 minutes

8. Wash with CHCl ₃ twice for 2 minutes

9. Wash with CH ₂ Cl ₂ three times for 2 minutes

10. Addition of Boc-amino acid -

11. Coupling with DIC once 120-300Mi luten

12. Wash with CH ₂ Cl ₂ twice for 2 minutes

13. Wash with ethanol

The cleavage of the Boc protecting group uses as scavenger a mixture of 0.5% indole and 0.2% thioanisole or 1% anisole and 0.5% diethyl sulfide.

In the synthesis, calculated on 1 millimole of Boc-Gaba-BHA resin, 3 millimoles of Boc-amino acid, 3 millimoles of DIC coupling agent and 3 millimoles of HOBt catalyst are used in a sequence corresponding to the sequence in a solution formed with dichloromethane. In the coupling of Boc-Arg (Tos) OH, a 1: 1 mixture (by volume) of dimethylformamide and dichloromethane is used. The coupling of all protected amino acids is carried out by the carbodiimide method. However, in the coupling of Boc-Gln and Boc-Asn, respectively, the p-nitro-phenyl ester of the corresponding protected amino acid is used in a threefold excess in the presence of an equivalent amount of the HOBt catalyst. In this case, the reaction is carried out in a 1: 1 mixture (by volume) of dimethylformamide and dichloromethane for 12-14 hours. The side chains of the protected amino acids are protected in the case of Tyr, Ser and Thr with the benzyl group, in Lys with the 2-chloro-benzyloxycarbonyl group (2 Cl-Z), in the case of Arg with the tosyl group (Tos), and the carboxyl groups of GIu and Asp are protected with the cyclohexyl group (Chx). The side chain protecting groups and the peptide are removed from the resin by liquid HF so that the peptide-resin compound - in the range of 0.25 millimoles of peptide BHA resin - in the presence of 2.5 ml, 10 wt% Anisole containing p-cresol and 100 mg of dithiothreitol in about 2O-25 ml of HF at ⁰ ° C. for one hour. The HF is removed under reduced pressure. The residue is treated with absolute diethyl ether, after which the peptide is detached from the solid residue with 15% by weight acetic acid. The peptide is purified in 15% by weight acetic acid on a Sephadex G-50 column by gel filtration. Thereafter, the peptide is by the MPLC method on a C ₁₈ reverse phase silica gel (Syncroprep RP-4 3 u) after a gradient elution using from 0.25 M triethylamine phosphate (TEAP, pH: 2.557) and 40 vol .-% TEAP -60Vol .-% acetonitrile prepared solutions. The fractions are identified by TLC and HPLC analysis. The fractions containing the desired product are combined and the solvent is removed under reduced pressure. The residue is first salted free and then returned to an MPLC column for further purification and subjected to gradient elution with a 10% by weight acetic acid solution containing 10% by weight of acetic acid solution and 30% by volume of isopropanol. The purity and product content of the fractions

are determined in the above manner. The peptide is finally separated by freeze-drying in the form of a solid substance.

The characteristic physical constants of the product are as follows:

TLC: Rf = 0.40 (Adsorbent: DC Alufoil Kieselgel 60 (catalog no. 55: 3), eluent: 4: 1: 2 (parts by volume) mixture of n-butanol: acetic acid: water)

HPLC: The adsorbent used is a Vydac 218 TP546 column and the eluant used is a mixture of 30% by volume of acetonitrile and 70% by volume of a 0.25M aqueous triethylammonium phosphate solution (pH 2.257). The determination is carried out on the basis of UV absorption at 215 nm and a sensitivity of 0.1 AuFS. The ISCO (Model 2350) HPLC pumps are controlled by an IBM-XT computer according to an ISCO ChemResearch program. An isochratic elution is carried out. The product is characterized by the ratio of retention time to breakthrough time (k '(k' = 4.37.

Example 2 Preparation of Nle ", Gaba ³⁰ -hGRF (1-3O) -OH

The peptide is prepared according to Example 1, but on a Morrifiuld resin (chloromethyl resin). The resin (capacity:

0.5-0.9 meq / g) is reacted with a threefold excess of Boc-Gaba cesium salt, in dimethylformamide, at 50 ° C for 20 hours As a result of the reaction, a 0.4-0.6 millimole Gaba / g substituted resin.

The peptide synthesis is carried out in the manner described in Example 1. The peptide obtained is, as specified in Example 1, purified. After TLC and HPLC analysis, the product obtained is uniform.

Example 3 Preparation of D-Ala ² , Nle ", Gaba ³⁰ -hGRF (1-30) -NH ₂

The procedure is as in Example 1, with the difference that the protected amino acid Boc-D-Ala is used as the second member of the peptide instead of Boc-Ala. The physical constants of the product are determined as described in Example 1. After TLC and HPLC analysis, the product is uniform.

TLC: R, = 0.39

HPLC: k '= 4.55.

Example 4 Preparation of D-Ala ² .Leu ^l5, Nle ', Gaba ³⁰ -hGRF (1-30) -NH ₂

The procedure is as in Example 3, with the difference that the protected amino acid Boc-Leu is used as the 15th member of the peptide instead of Boc-Gly. The physical constants of the product are determined as described in Example 1. After TLC and HPLC analysis, the product is uniform.

TLC: R, = 0.41

HPLC: k '= 4.55.

Example 5 Preparation of D-Ala ^I , D.Arg ^1l _l Leu ' ^s , Nle ", Gaba ³⁰ -hGRF (1-30) NH ₂

The procedure is as in Example 3, with the difference that at the 11-position of the peptide chain instead of Boc-Arg (Tos) the protected amino acid Boc-D-Arg (Tos) and protected at the 15-position instead of Boc-Gly Amino acid Boc-Leu couples. The physical constants of the product are determined as described in Example 1. After TLC and HPLC analysis, the product has proved to be uniform.

TLC: R, = 0.38

HPLC: k '= 3.52.

Example 6

Preparation of D-Ala ^2, Phe ^'s, Nle', Gaba ³⁰ -hGRF (1-30) -NHj

The procedure is as in Example 3, with the difference that at the 15-position of the peptide chain instead of Boc-Gly, the protected amino acid Boc-Phe couples. The physical constants of the product are determined as described in Example 1.

After TLC and HPLC analysis, the product is uniform.

TLC: R, = 0.42

HPLC: k '= 4.75.

Example 7

Preparation of D-Ala ² , desGly ' ⁶ , Nle ", Gaba ³⁰ -hGRF (1-30) -OH

The procedure is as in Example 2, with the difference that at the 2-position of the peptide chain instead of Boc-Ala the protected amino acid Boc-D-Ala couples, and in the 15-position of the peptide chain, the coupling with Boc-Gly is omitted , The physical constants of the product are determined in the manner described in Example 1. After TLC and HPLC analysis, the product is uniform.

HPLC: k '= 4.40.

Example 8

Preparation of D-Ala ² , teu ¹⁵ , Nle ²⁷ , Gaba ³⁰ -hGRF (1-30) -OH

The procedure is as in Example 7, with the difference that in the 15-position of the peptide chain Boc-Leu is coupled. The physicati '.en constants of the product are determined av' the manner described in Example 1. After TLC and HPLC analysis, the product is uniform.

HPLC: k '= 4.60.

Example 9 Production of Gaba ³⁰ -bGRF (1-30) -NH ₂

The procedure is as in Example 1, with the difference that at the 27-position of the peptide chain instead of Boc-Nle the protected amino acid Boc-Met and at the 28-position instead of Boc-Ser (bzl) dor active Boc-aspartic acid ester is coupled. After TLC and HPLC analysis, the product is uniform.

Example 10 Preparation of Nte ", Gaba ³⁰ -bGRF (1-3O) -NH ₂

The procedure is as in Example 9, with the difference that is coupled at the 27-position of the peptide chain Boc-Nle. After TLC and HPLC analysis, the product is uniform.

Example 11 Production of Gaba ³⁰ -oGRF (1-30) -NH ₂

The procedure is as in Example 9, with the difference that in the 13-position of the peptide chain instead of Boc-Val the protected amino acid Boc-Ile couples. After TLC and HPLC analysis, the product is uniform.

Example 12 Preparation of Nle ", Gaba ³⁰ -oGRF (1 -3O) -NH ₂

The procedure is as in Example 11, with the difference that one couples at the 27-position of the peptide chain instead of Boc-Met Boc-Nle, after TLC and HPLC analysis, the product is uniform.

Claims (1)

claims: 1. A process for the preparation of novel peptides of the general formula (I) and salts thereof H-Tyr-R ² -Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr -R ¹¹ -Lys-R ¹³ -Leu-R ¹⁵ -Gln-Leu-Ser-Ala-Arg- (I) -Lys-Leu-Leu-Gln-Asp-Ile-R "-R ²⁸ -Arg-Gaba-Y, wherein' R ^{2 is} Ala or D-Ala, R ^{11 is} Arg or D-Arg, R ^{13 is} VaI or He, R ^{15 is} Gly, Leu, Phe or a valence bond, R "mead or never means R ²⁸ Ser, D-Seroder Asnist, and Y is OH or NH ₂ ,