CS276972B6 - Peptides, process of their preparation and pharmaceutical containing thereof - Google Patents

Abstract

Synthetic peptides of formula (I) and their nontoxic salts are new. R1-R2-R3-Ala- Ile-Phe-Thr-R8- Ser-R10-Arg-R12-R13-Leu-R13-Gln- R17-R18-Ala-Arg- Lys-Leu-R23-R24- R25-Ile-R27-R28-Arg-Gln-Gln-Gly- Glu-R34-Asn-Gln- Glu-R38-R39-R40-Arg-R42-R43-R44 (I) In (I), R1=Tyr, D-Tyr, Met,Phe, D-Phe, 4-chloro-Phe, Leu, His or D-His, and is opt. C alpha- or Nalpha-CH3 substd. R2 = Ala or D-Ala; R3 = Asp or D-Asp; R8 = Ser, Asn, D-Ser or D-Asn; R10= Tyr or D-Tyr; R12 = Arg or Lys; R13= Ile or Val, R15 = Gly or D-Ala; R17 = Leu or D-Leu; R18 = Tyr or Ser; R23 = Leu or D-Geu; R24 = His or Gln; R25 = Glu, Asp, D-Gln or D-Asp; R27 = Met, D-Met, Ala, Nla, Ile, Leu, Nva or Val; R28 = Asn or Ser; R34 = Arg or Ser; R38 = Gln or Arg; R39 = Arg or Gly; R40 = Ser or Ala; R42 = Phe, Ala or Val; R43 = Asn or Arg; R44 = natural amino, and any or all of residues R29 to R44 can be deleted. Proviso are of the following conditions must apply: (I) R1 is alpha-CH3 substd; (2) R17 and/or R23 is D-Leu; (3) R25 is D-Glu or D-Asp.

Description

Field of the Invention

The invention relates to peptides that affect pituitary function in humans and other animals. In particular, they are peptides that promote the release of growth hormone from the pituitary. The invention also relates to a process for the preparation of these peptides and pharmaceutical compositions containing them.

BACKGROUND OF THE INVENTION

Physiologists have long known that the hypothalamus controls the secretory function of adenohypophysis by producing special substances that promote or inhibit the secretion of individual pituitary hormones. In 1982, growth hormone releasing factor (hpGRF) was isolated from human pancreatic tumor extracts, purified, characterized, synthesized and tested, and was shown to promote the release of growth hormone by the pituitary gland. In addition, other similar factors were synthesized and characterized, namely rat growth hormone releasing factor (rGRF) and corresponding swine factor (pGRF).

New polypeptides that release growth hormone from pituitary flasks grown in tissue culture have now been synthesized, these peptides at least partially resisting degradation of the enzyme in an animal organism. Said peptides meet at least one of the following requirements:

(a) the residue at position 1 is substituted either by the method N ^{a1 and} Me, or C ^{a1 and} Me,

(b) D-Leu is in position 17 and / or position 23 and / or

c) D-Glu or D-Asp is in position 25.

Peptides may meet a plurality of these requirements, but at least one of them must meet these requirements. The residue at position 1 may be Tyr, D-Tyr, Met, Phe, D-Phe, pCl-Phe, Leu, His and D-His (this residue may carry a methyl group either at the alpha position on the carbon atom or on the alpha- amino). The peptides usually comprise D-alpha at position 2 and / or D-Asp at position 3 and / or D-Arg or D-Sex at position 8 and / or D-Tyr at position 10 and / or D-Alpha at position

15. Preferably, the peptides are also substituted with D-Met, Nle or Nve instead of Met at position 27.

The pharmaceutical compositions may comprise analogs produced by the process of the invention of 28 to 44 amino acid residues in length or a non-toxic salt thereof in the form of a dispersion in a pharmaceutically acceptable veterinary or solid carrier. The composition thus obtained can be used in clinical medicine, in human and veterinary medicine, for therapeutic and diagnostic purposes. The compounds can be used to accelerate the growth of warm-blooded animals including poultry and in agriculture as well as to accelerate the growth of cold-blooded animals such as fish and the like.

The nomenclature for novel peptides was used according to Schroder and Lubke The Peptides, Academic Press (1965) such that, according to common nomenclature, the amino group at the N-terminal end is on the left and the carboxyl group at the C-terminal end is on the right side. For natural amino acids, these are naturally occurring substances found in proteins, such as Gly, Ala, Val, Leu, Ile, Ser, Thr, Lys, Arg, Asp, Asn, Glu, Gin, Cys, Met, Phe, Tyr, Pro, Trp, and His. Nle is norleucine and Nva is norvaline. Where the amino acid residue has isomeric forms, the L-form is always understood unless otherwise indicated.

SUMMARY OF THE INVENTION

The present invention relates to peptides of the general formula I ^R 1 ^R 2 ' ^R 3 " ^Ala " ^Ile " ^Rhe " ^Thr " ^R 8 ~ ^Ser " ^R 10 ^Ar ^ ^R 12 " ^R 13" ^Leu " ^R 15" ^Gln " ^R 17" Rig -Ala-Arg-Lys-LeU "R23R24R25" ^Ile " ^R 27 ^R 28" ^Ar g ^_Gln " ^Gln '* ^G1 Y" ^Glu · Rg 4 —Asn — Gin — Glu — R3 θ -R ₃ g “^ 4 o Arg —R _{4 2} “^ 4 3“ ^ 4 4 (I) where

^R 1 means Tyr, D-Tyr, Met, Phe, D-Phe, pCl-Phe, Leu, His or D-His, in substitution without substitution, type _c alpha ^and Me or N ^alpha Me or ^R 2 means Ala or D-Ala, ^R 3 means Asp or D-Asp, R _g means Ser, Asn, D-Ser, or D-Asn, ^R 10 means Tyr or D-Tyr, ^R 12 is Arg or Lys, ^R 13 means Ile or Val, ^R 15 means Gly or D-Alpha, ^R 17 means Leu or D-Leu, ^R 18 means Tyr or Ser, ^R 23 means Leu or D-Leu, ^R 24 means His or Gin, ^R 25 means Glu, Asp, D-Glu or D-Asp, ^R 27 means Met, D-Met, Ala, Nle, Ile, Leu, Nva, or Val,

^R 28 means Asn or Ser, ^R 34 means Arg or Ser, ^R 38 means Gin or Arg, R39 means Arg or Gly, ^R 40 means Ser or Ala, ^R 42 means Phe , Ala or Val ^R 43 means Asn or Arg, ^R 44 means Wall or Leu,

provided either

a) Rj. has a substitution type ^{C, and Me-Al} or ^{Al-N and} Me or

b) R ₁₇ and / or R ₂₃ represents D-Leu;

c) R ₂₅ is D-Glu or D-Asp, and any one of ^R 44 may be omitted ^;

^R 43 ^R 44 ' ^R 42 ^-R 43 ~ ^R 44'

Arg- ^R 4 ₂ -R ₄ 3- ^ 44 /

R40 “Arg“ ^R 42 “ ^R 43“ ^R 44z ^R 39 “ ^R 40 ^{Arg R} 42 ™ ^R 43” ^R 44 ' ^R 3 8 “ ^R 39” ^R 4 0 ” ^Ar 9” ^R 42 ” ^R 4 3 ~ ^R 4 4 '

Glu-R ₃ gR ₃ 9 'R ₄ Q-Arg-R ₄₂ -R ₄₃ -R ₄₄

Gln-Glu-R ₃ g 'R ₃ 9R 4 o-Arg-R _{4 2} -R _{4 3} -R _{4 4} ,

Asn — Gln-Glu-R ₃ θ-R _{3 g} -R ₄ θ-Arg-R4 ₂ -R _{4 3} -R _{4 4} , ^R 3 4 “Asn-Gln-GlU” R ₃ θ “ ^R 3 9” ^R 4 0 “ ^Ar 1 ^R 42” ^R 43 “ ^R 44 ·

G1u-R _{3 4} -Asn-Gln-Glu-R ₃ gR ₃ gR ₄ Q-Arg-R _{4 2} ~ R _{4 3} 'R ₄₄ ,

Gly-Glu-R _{3 4} -Asn-Gln-Glu-R _{3 8} -R _{3 g} -R _{4 0} -Arg-R _{4 2} -R ₄₃ "R44, Gln-Gly-Glu-R _{3 4} -Asn-Gln -Glu-R _{3 g} -R _{3 g} -R ₄ θ-Arg-R _{4 2} -R _{4 3} -R _{4 4} , Gln-Gln-Gly-Glu-R _{3 4} -Asn-Gln-Glu-R _{3 8} -R _3g -R _{4 Q} -Arg-R _{4 2} -R ₄₃ -R _{4 4} ·

The invention also relates to a process for the production of these peptides which comprises:

i) binds the N-terminal amino acid protected with a tert-butoxycarbonyl group to an -NH- benzhydrylamine resin; or ii) a protective target. the butoxycarbonyl group cleaves and sequentially bound individual amino acids in an amount of 1 to 2 mmol of amino acid protected by the target. butoxycarbonyl group, per gram of resin in methylene chloride, dimethylformamide or a mixture of these solvents to give a compound of formula II with at least one protecting group

X ^lm RL (X or X ² -R 2 -R ₃ (X ³⁾ -Ala-Ile-Phe-Thr (R ⁴⁾ -R 8 (X ⁴ or X ⁵⁾ -Ser (X ⁴⁾ -R 10 (X ²⁾ -Arg (X ⁶⁾ -R 12 (X ⁶ or X ⁷⁾ -R 13 -Leu-R ₁₅ -Gln (X ^{5) 'r} 17 ^r 18 ^{(χ2)' Ala} 'Arg (X ⁶⁾ -Lys (X ⁷⁾ -Leu-R23-R24- (X or X ⁵⁾ -R 25 (X ³⁾ -Ile-R27-R2g (X ⁴ or X ⁵⁾ -Arg (X ⁶⁾ -Gln (X ⁵⁾ -Gln (X ⁵⁾ -Gly-Glu ^(X3) -R34 (X ⁴ or X ⁶⁾ -Asn (X ⁵⁾ -Gln (X ⁵⁾ -Glu (X ³⁾ -R3g (X ⁵ or X ⁶⁾ -R 39 (X ⁶⁾ -R ₄₀ (X ⁴ ) -Arg (X ⁶ ) -R ₄₂ -R ₄₃ (X ⁵ or X ⁶ ) -R ₄₄ -NH-resin, (II) wherein

X ¹ represents a hydrogen atom or a protecting group on the - -amino group,

X ² represents a hydrogen atom to the hydropolymer Tyr, or protective group to phenolic X ³ represents a hydrogen atom with Asp or Glu, or a carboxyl protecting group X ⁴ represents a hydrogen atom to the hydro group Thr, or protective group to alcohol X ⁵ represents a hydrogen atom for the Ser radical, or protective group to alcohol X ⁶ represents a hydrogen atom or guanidine protecting group

the Arg group,

X ⁷ represents a hydrogen atom or a protecting group on the amino group of the Lys side chain,

X represents a hydrogen atom or a protecting group on the nitrogen atom of His imidazole group, iii) cleavage of the protecting group or protecting groups to give compounds of formula II to form a peptide of formula I or a biologically active fragment thereof and optionally iv) converting the resulting peptide into its nontoxic addition salt.

Further, the possible and advantageous meanings of the individual protecting groups will be discussed.

X ¹ represents a hydrogen atom or a protecting group on a X X -amino group. These are protecting groups commonly used in the sequential synthesis of polypeptides. The protecting groups X ¹ can be classified into several groups, namely

1) urethane-type aromatic protecting groups, for example fluorenylmethyloxycarbonyl (FMOC), benzyloxycarbonyl (Z) and substituted Z, for example p-chlorobenzyloxycarbonyl, p-nitrotrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl and p-methoxybenzyloxycarbonyl,

2) aliphatic urethane-type protecting groups, e.g. butyloxycarbonyl (BOC), diisopropylmethyloxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, allyloxycarbonyl and

3) cycloalkylurethane-type protecting groups such as cyclopentyloxycarbonyl, adamantyloxycarbonyl and cyclohexyloxycarbonyl;

A preferred protecting group on the -amino group is BOC, even when an N * Me-substituted residue is used in the 1-position. -X represents a hydrogen atom or a protecting group for the imidazole nitrogen atom of the amino acid His, for example Tos.

X ² is a protecting group for the phenol hydroxyl group Tyr, for example tetrahydropyranyl, tert. butyl, trityl, Bzl, CBZ, 4br-CBZ and 2,6-dichlorobenzyl (DCB). A preferred protecting group is 2,6-dichlorobenzyl; X ² may also wipe hydrogen, meaning that there is no protecting group at the amino acid residue at this position.

X ³ represents a hydrogen atom or a suitable ester-forming group with the carboxyl group Asp or Glu, for example benzyl (OBzl), 2,6-dichlorobenzyl, methyl and ethyl.

X ⁴ is a suitable protecting group for the hydroxyl group Thr or Ser, for example acetyl, benzoyl, tert-butyl, trityl, tetrabvdropyranyl, Bzl, 2,6-dichlorobenzyl and CBZ. A preferred protecting group is Bzl. X ⁴ may also be hydrogen, in which case there is no protecting group on the hydroxyl group.

X ⁵ represents a hydrogen atom or a suitable amide side chain protecting group, Asn or Gln. It is preferably xanthyl (Xan).

X ⁶ is a suitable protecting group for the guanidino group. Arg, for example, nitro, Tos, CBZ, adamantyloxycarbonyl and BOC, or is hydrogen.

X ⁷ is hydrogen or a suitable amino protecting group for Lys. Suitable amino protecting groups are, for example, 2-chlorobenzyloxycarbonyl (2-C1-Z), Tos, t-amyloxycarbonyl and BOC.

X ⁸ represents a hydrogen atom or a suitable side chain protecting group as defined above.

The met may optionally be protected by an oxygen atom, but is preferably not protected.

The choice of side chain protecting groups is not critical, except that a group that is not removed during deprotection at the α-amino group in the synthesis is required. For some amino acids, such as His, it is not usually necessary to use a protecting group after the binding has been completed or it may be the same protecting group.

X ⁹ represents a suitable protecting group on a C-terminal carboxyl group, for example X ³ , forming an ester or is an anchoring bond in solid-phase synthesis to bind to a resin as a solid support, or is des-X ⁹ , In this case, the residue at the C-terminal end has a carboxyl group Y as mentioned above. When used as a solid resin support, this resin is also broadly considered as a protecting group, thus forming a group of -O-CH2-resins as support, -NH-benzhydrylamine (BHA) resin as support, or -NH- Paramethylbenzhydrylamine (MBHA) resin as a support.

If an unsubstituted amide is desired, the use of BHA or MBHA resin is preferred because cleavage results directly in the amide. When N-methylamide is desired, it can be obtained from N-methyl BHA resin. When substituted amides or groups other than the free acid at the C-terminal end are desired, it may be advantageous to synthesize the peptide by the classical method as described in Houben-Weyl.

In the formula of the intermediate, at least one X group is a protecting group or the X group ⁹ comprises a resin as a support. Thus, by the method of the invention, it is possible to obtain the peptide by:

a) forms an intermediate peptide with at least one protecting group of formula II:

X ¹ -R ¹ (X or X ² ) -R ² -R ₃ (X ₃ ) -Ala-Ile-Phe-Thr (X ⁴ ) -R 8 (X ⁴ or

X ⁵⁾ -Ser (X ⁴⁾ -Tyr (X ²⁾ -Arg (X ⁶⁾ -R 12 (X ⁶ or X ⁷⁾ -R 13 -Leu-Gly-Gln (X ⁵⁾ -R X 7 -R 8-j (X ²⁾ -Ala-Arg (X ⁶⁾ -Lys (X ⁷⁾ -Leu-R 2 -R 3 _{2 5} (X ⁵ or X) -R 25 (X ³⁾ -Ile-R 27 -R ₂₃ (X ⁴ or X ⁵⁾ -Arg (X ⁶⁾ -Gln (X ⁵⁾ -Gln (X ⁵⁾ -Gly-Glu ^(X3) -R34 (X ⁴ or X ⁶⁾ -Asn (X ⁵⁾ -Gln (X ⁵⁾ -Glu (X ³⁾ -R 38 (X ⁵ or X ⁶⁾ -R 39 (X ⁶⁾ -R 40 (X ⁴⁾ -Arg- (X ⁶⁾ -R ₄₂ -R ₄₃ (X ⁵ or X ⁶⁾ 7

-r ₄₄ (x ⁸ ) -x ⁹ where X, X ¹ , X ² , X ³ , X ⁴ , X ⁵ , X ⁶ , X ⁷ and X ⁸ represent a hydrogen atom or a protecting group and X ⁹ represents a protecting group or an anchoring agent a resin group as a support or des-X ⁹ , in which case the residue at the C-terminal end is a carboxyl group Y,

(b) cleavage of the protecting group, protecting groups or anchoring bond of the peptides of formula (II); and

c) optionally converting the resulting peptide into one of its non-toxic salts.

The following general rules should be followed when choosing a particular side chain protecting group for peptide synthesis:

(a) the protecting group should retain its protective properties and should not be cleaved off at the bonding of the next residue;

(b) the protecting group should be stable to the reagents used and, except for Xan, preferably stable under the reaction conditions selected to remove the protecting group on the x, -amino group at each stage of the process; and

(c) the side protecting groups must be capable of being removed after completion of the process to yield the resulting peptides containing the desired amino acid sequence so that undesired changes in the peptide chain do not occur during removal.

When peptides are not prepared by recombinant DNA technology, they are preferably prepared using solid phase synthesis, for example, according to the method of Merrifield, J. Am. CHem. Soc., 85, 2149 (1963), although other equivalent chemical methods as described above may also be used.

Solid phase synthesis starts from the C-terminal end of the peptides by binding the protected α-amino acid to a suitable resin. This starting material can be prepared by coupling the protected amino acid via an ester bond with a chloromethylated resin or a hydroxymethyl resin or an amide bond to a BHA resin or an MBHA resin. The preparation of hydroxymethyl resin is described in Bodansky et al., CHem. Indian. (London) 38, 1597-98 (1966). Chloromethylated resins are commercially available (Bio Rad Laboratories, Richmond, California and Lab. Systems, Inc). The production of a resin of this type is described in Stewart et al., Solid Phase Peptide Synthesis, Freeman and Co., San Francisco 1969, Chapter 1, pages 1-6. BHA and MBHA resins are also commercially available and are usually used only in this case. that the desired peptide is to be synthesized as an unsubstituted amide at the C-terminal end.

The C-terminal amino acid, i.e., Asn, protected by BOC and Xan, can first be coupled to a chloromethylated resin by the method of Chemistry Letters, K. Horiki et al., 165-168 (1978), using KF in dimethylformamide at 60 ° C. for 24 hours with stirring to obtain a 43 residue peptide. After the BOC-protected amino acid has been attached to the resin as a support, the amino-protecting group is removed, for example using trifluoroacetic acid (TFA) in methylene chloride or trifluoroacetic acid alone. The deprotection is carried out at room temperature. Other conventional reagents such as hydrochloric acid in dioxane may also be used for cleavage using conditions specific for the removal of the individual protecting groups on the .alpha. Group as described in Schroder and Lubke, The Peptides, 1, p. 72-75 (Academic Press 1965) ·

After removal of the X-amino protecting group, the remaining amino-amino and side chain protected amino acids are sequentially coupled in the desired sequence to form the above intermediate, or it is also possible to link some amino acids to each other before adding the entire shorter chain to bound parts on the solid phase. The selection of the appropriate reagent can be made by any person skilled in the art. A particularly suitable reagent for use herein is N, N'-dicyclohexylcarbodiimide (DCCI).

The activating agent used in solid phase synthesis may be selected from well known reagents in peptide chemistry. Examples of suitable activating reagents are carbodiimides such as Ν, Ν'-diisopropylcarbodiimide and N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide. Other activating reagents may also be used, and these reagents and their use in peptide binding have been described in Schroder and Lubke, supra, Chapter III, and in Kapoor, J.Phar. Sci. 59 pages 1-27 (1970).

Each protected amino acid or sequence of amino acids is fed to the solid phase, in a four-fold or greater excess, in a 1: 1 mixture of dimethylformamide and methylene dichloride or dimethylformamide or methylene dichloride alone. In the case of incomplete coupling, the procedure is repeated before the deprotection of the α-amino group and before the subsequent amino group is attached. The success of the binding at each stage of the synthesis, when performed manually, is preferably verified by a ninhydrin reaction as described by E. Kaiser et al., Anal. Biochem. 34, 595 (1970). Coupling can also be carried out automatically, for example by an automatic synthesizer (Beckman 990), as described in Rivier et al., Biopolymers, 1978, 17, pages 1927-1938.

After gaining complete the desired amino acid sequence, the peptide can be obtained as an intermediate product removed from the resin as the substrate with an agent, such as liquid hydrogen fluoride, which not only cleaves the peptide from the resin but also cleaves all the remaining protecting groups on the side chain X, X ^2, X ³ , X ⁴ ,

X ⁵ , X ⁶ , X ⁷ and X ⁸ and X ⁸ and X ⁹ as the anchoring bond, as well as the protecting group X ¹ on the α-amino group, if used, to give the resulting peptide as the free acid. If Met is present in sequence, preferably the BOC protecting group is first removed using a mixture of trifluoroacetic acid and ethanedithiol prior to cleavage of the peptides from the resin by hydrofluoric acid to eliminate potential S-alkylation. When hydrogen fluoride is used for the cleavage, the reaction mixture is supplemented with anisole and methyl ethyl sulfide as substances which bind the by-products.

The invention will be illustrated by the following examples which describe preferred methods of carrying out the solid phase method to obtain the resulting peptides. It goes without saying that the synthesis of the corresponding shorter peptide fragment is carried out in the same manner by simply omitting the corresponding amino acids, but it is likely that the biologically active fragments should contain the above sequence adjacent to the N-terminal end.

Example 1

Synthesis of peptides of formula [N ⁰⁶ MeHis ¹ ] / rhGRF- (1-43) -0H, detailed in detail:

N-MeHis-Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Arg-Ile-Leu-Gly-Gln-Leu-Tyr-Ala-Arg-Lys-Leu-Leu-His- Glu-Ile-Met-Asn-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln-Gln-Gln-Arg-Ser-Arg-Phe-Asn-OH is performed sequentially using a peptide synthesizer (Beckman 990) on a chloromethylated resin with a substitution of about 0.1 to 0.5 mmol / g resin. Binding of BOC-Asn (Xan) to the resin was carried out as described in Chemistry Letters above using potassium fluoride in dimethylformamide at about 60 ° C for 24 hours with stirring, resulting in a substitution of about 0.35 mmol Asn to gram resin.

After deprotection and neutralization, the peptide chain is gradually built on the resin. Deprotection, neutralization, and addition of each additional amino acid are performed as described in Rivier J. J. Amer. Chem. Soc., 96, 2986-2992 (1974). All solvents used are carefully degassed by flushing with an inert gas such as helium or nitrogen to ensure there is no oxygen in which undesirable oxidation of sulfur in the Met residue may occur.

The deprotection is preferably carried out according to the following scheme A.

Scheme A

Reagents Mixing time (min)

1. 60% trifluoroacetic acid 10 with 2% ethanedithiol

2. 60% trifluoroacetic acid 15 with 2% ethanedithiol

3. IPA with 1% ethanedithiol 0.5

4. 10% Et ₃ N in methylene dichloride 0.5

5. methanol 0.5 6. 10% Et ₃ N in methylene dichloride 0.5 7. Methanol (twice) 0.5 8. methylene dichloride (twice) 0.5 Binding of other amino acids of Scheme B: is carried out according to the following Scheme B Reagents Mixing time (min.) 9. DCCI .. - 10. Boc-amino acid 50 to 90 11. methanol (twice) 0.5 12. methylene dichloride (twice) 0.5 13. 3M Ac ₂ O in methylene dichloride 15.0 14. methylene dichloride 0.5 15. methanol 0.5 16. methylene dichloride (twice) 0.5 1 to 2 mmol of BOC-protected methylene- chloride per g resin; and in addition, 1 equivalent of 1.0 OM DCCI. After BOC-Arg (TOS) was bound in methylene chloride for 2 hours a mixture of 50% dimethylformamide < and 50% methylene chloride. Bzl ve

The ether form is used as a protecting group for the hydroxyl group of the side chains Ser and Thr. The amide group Asn or Gln is protected by Xan when dicyclohexylcarbodiimide is used as the coupling agent. It is also possible to use P-nitrophenyl ester (ONp) to activate the carboxyl terminus of Asn or Gln, and for example BOC-ASn (ONp) can be coupled overnight using one equivalent of HOBt in 50% dimethylformamide / methylene chloride. . 2-chlorobenzyloxycarbonyl (2Cl-Z) is used as a Lys side chain protecting group. TOS is used to protect the guanidine group of Arg and to protect the nitrogen atom of the imidazole residue His, and the carboxyl group of the Glu or Asp side chain is protected by OBzl. The phenol hydroxyl group of Tyr is protected by a 2,6-dichlorobenzyl group (DCB). At the end of the synthesis, the following compound is obtained:

BOC-N * MeHis (X) -Ala-Asp (X ³⁾ -Ala-Ile-Phe-Thr (X ⁴⁾ -Ser (X ⁴⁾ -Ser (X ⁴⁾ -Tyr (X ²⁾ -Arg (X ⁶⁾ -Arg ^(X6) -Ile-Leu-Gly-Gln ^(X5) -Leu-Tyr ^(X2) -Ala-Arg (X ⁶⁾ -Lys (X ⁷⁾ -Leu-Leu-His (X ) -Glu (X ³⁾ -Ile-Met-Asn (X ⁵⁾ -Arg (X ⁶⁾ 11

-Gin (X ⁵⁾ -Gin (X ⁵⁾ -Gly-Glu (X ³⁾ -Arg (X ⁶⁾ -Asn (X ⁵⁾ -Gin (X ⁵⁾ -Glu (X ³⁾ -Gin (X ⁵⁾ -Arg ^(X6) -Ser ^(X4) -Arg ^(X6) -Phe-Asn (X ⁵⁾ -X ⁹ wherein

X '

X '

X '

X means means means means means

It with,

DCB

Obzl,

BZl,

Xan,

It with

2C1-Z a

_-O-CH2 -resin padding.

Xane can be partially or completely removed by treatment with trifluoroacetic acid, which is used to remove the protecting group on C <. -aminogroup.

A mixture of 1.5 ml of anisole, 0.5 ml of methylethylsulfide and 15 ml of hydrofluoric acid per g of peptide-resin complex is used to cleave the peptide from the resin and remove the protecting groups at -20 ° C for 1/2 hour and then at temperature 0 ° C 1/2 hour. After removal of the hydrogen fluoride under high vacuum, the resin and peptide residue are washed alternately with anhydrous diethyl ether and chloroform, and then the peptide is extracted with a degassed 2N aqueous acetic acid solution and separated from the resin by filtration.

The cleaved, deprotected peptide is then dissolved in 0 to 5% acetic acid and purified, for example, by Sephadex G-50 gel filtration.

Thereafter, the peptide is further purified by preparative or semi-preparative high pressure liquid chromatography according to the method of Revivier et al., Peptides: Structure and Biological Function (1979), pages 125-128; and Marki et al., J. Am. CHem. Soc. 103, 3178 (1981). Column (Waters Associates prep LC-500) is filled with 15 to 20 C for ₁₈ -kysličníkem silica (Vydac 300A). The CH ₃ CN gradient in TEAP is generated by the apparatus (Eldex) as described by Rivier J., J. Lig. CHromatography 1, 343-367 (1978). Chromatographic fractions are carefully monitored by high pressure liquid chromatography and only fractions of sufficient purity are combined. The removal of salts from the purified fractions is achieved using a gradient of CH ₃ CN in 0.1% trifluoroacetic acid. The middle portion is then lyophilized to give the desired peptide, which may be greater than 98% pure.

The synthesis was repeated using MBHA resin to give the same peptide with an amidated C-terminal end, following the procedure described in Vale et al., U.S. Patent 4,292,313 to bind Asn to MBHA resin.

Example 2

Synthesis of 40 residue peptides of formula HC * MeHis-Ala-Asp-Ala-Ile-Phe-Thr-Ser-Tyr-Arg-Arg-Arg-Ile-Leu-GLy-Gln-Leu-Tyr-Ala-Arg-Lys-Leu -Leu-His-Glu-Ile-Met-Asn-Arg-Gln-Gln-Glu-Arg-Asn-Gln-Glu-Gln-Arg-Scer-NHg is performed sequentially using a peptide synthesizer (Beckman 990) and MBHA resin by the method generally described by Val et al., U.S. Pat. No. 4,292,313. Thin layer chromatography and high pressure liquid chromatography showed that substantially pure peptide was obtained.

Example 3

Synthesis of rhGRF analogue, ie:

[N, Me-Met ¹ , Leu ²⁷ ] -rhGRF (1-43) OH of formula

N-MeMet-Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Arg-Ile-Leu-Gly-Gln-Leu-Tyr-Ala-Arg-Lys-Leu-Leu-His- Glu-Ile-Leu-Asn-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln-Glu-Gln-Arg-Ser-Arg-Phe-Asn-OH is performed sequentially using a peptide synthesizer (Beckman 990) and chloromethylated resins in the manner generally described in Example 1. Depending on the results of thin layer and high pressure liquid chromatography, essentially pure peptide is obtained.

Example 4

Synthesis of the hpGRF analog fragment, i.

[C MePhe (4Cl) ¹ ] -hpGRF (1-32) -NH _{2 of the} formula

HC MePhe (4Cl) -Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu- _{Gln-Asp-Ile-Met-Ser-Arg-Gln-Gln-Gly-NH2} is conducted sequentially with a peptide synthesizer (Beckman 990) on an MBHA resin as described in Example 2. the results of thin layer chromatography and high pressure liquid chromatography is essentially pure.

The synthesis was repeated two more times to give [4Cl-Phe ¹ ] -hpGRF (1-32) -NH 2 and [N * MePhe ¹ ] / hpGRF (1-32) -NH 2.

Example 5

Synthesis of the fragment of hpGRF analogue, ie [N ⁰⁶ MeTyr ¹ ] -hpGRF (1-29) -NH _{2 of} formula:

N MeTyr - Ala - Asp - Ala - Ile - Phe - Thr - Asn - Ser - Tyr - Arg - Lys - Val - Leu - Gly - Gln - Leu - Ser - Ala - Arg - Lys - Leu - Leu - Gln - Asp The Ile-Met-Ser-Arg-N @ 2 was carried out sequentially using a peptide synthesizer (Beckman 990) on MBHA resin as described in Example 2. Thin layer chromatography and high pressure liquid chromatography yielded substantially pure peptide.

Example 6

Synthesis of rhGRF fragment, i.

[N ⁰⁶ MePhe ¹ ] -rhGRF (1-29) -NH ₂ formulas:

N ⁰⁶ MePhe-Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Arg-Ile-Leu-Gly-Gln-Leu-Tyr-Ala-Arg-Lys-Leu-Leu-His- Glu-Ile-Met-Asn-Arg-NH ₂ was carried out sequentially using a peptide synthesizer (Beckman 990) on MBHA resin as described in Example 2. Thin layer chromatography and high pressure liquid chromatography yielded substantially pure peptide.

Example 7

Synthesis of [Ν '* MeLeu ¹ , Ile ²⁷ ] -hpGRF (1-32) -NH _{2 of} formula:

N ⁰⁶ MeLeu-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyl-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln- Asp-Ile-Ile-Ser-Arg-Gln-Gln-Gly-NH ₂ was carried out sequentially using a peptide synthesizer (Beckman 990) and MBHA resin according to the method of Example 2. According to the results of thin layer and high pressure liquid chromatography, yields a substantially pure peptide.

Example 8

Synthesis of rhGRF analog fragment, i.

[C * Me (4Cl) ¹ ] -rhGRF (1-29) -NH ₂ formulas:

HC MePhe (4Cl) -Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Ile-Leu-Gly-Gln-Leu-Tyr-Ala-Arg-Lys-Leu-Leu- His-Glu-Ile-Met-Asn-Arg-nh ₂ is carried out sequentially using a peptide synthesizer (Beckman 990) on MBHA resin as described in Example 2. Thin layer and high pressure liquid chromatography results in this manner. substantially pure peptide.

Example 9

Synthesis of rhGRF analog fragment, i.

[N ⁰⁰ MeTyr ¹ ] -rhGRF (1-29) -NH ₂ formulas:

HN ** MeTyr - Ala - Asp - Ala - Ile - Phe - Thr - Ser - Ser - Tyr - Arg - Arg - Ile - Leu - Gly - Gln - Leu - Tyr - Ala - Arg - Lys - Leu - His -Glu-Ile-Met-Asn-Arg-NH ₂ is carried out sequentially using a peptide synthesizer (Beckman 990) and MBHA resin as described in Example 2. Thin layer chromatography and high pressure liquid chromatography yield essentially the same. pure peptide.

Example 10

Synthesis of [C ^ MeLeu ¹ ] -rhGRF- [Val ⁴⁴ -NH ₂ ] formulas:

MeLeu-Ala-Asp-Ala-Ile-Phe-Thr-Ser-Tyr-Arg-Arg-Ile-Leu-Gly-Gln-Leu-Tyr-Ala-Arg-Lys-Leu-Leu-His-Glu -Ile-Met-Asn-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln-Glu-Gln-Arg-Ser-Arg-Phe-Asn-Val-NH ₂ is carried out sequentially using a peptide synthesizer on a resin MBHA according to the method described in Example 2. According to the results of thin layer chromatography and high pressure liquid chromatography, this method yields substantially pure peptide.

Example 11 -,

Peptide synthesis [Ν '* MeTyr ¹ , D-Le ²³ ] -rGRF (1-43) -OH of formula:

N ⁰⁶ MeTyr-Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Arg-Ile-Leu-Gly-Gln-Leu-Tyr-Ala-Arg-Lys-Leu-D-Leu- His-Glu-Ile-Met-Asn-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln-Glu-Gln-Arg-Ser-Arg-Phe-Asn-OH is performed sequentially using a peptide synthesizer (Beckman 990) on a chloromethylated resin with a substitution of about 0.1 to 0.5 mmol / g resin as described in Example 1.

At the end of the synthesis, a compound of formula:

BOC-N ⁰⁶ MeTyr ^(X2) -Ala-Asp (X ³⁾ -Ala-Ile-Phe-Thr (X ⁴⁾ -Ser (X ⁴⁾ -Ser (X ⁴⁾ -Tyr ^(X2) -Arg ( X ⁶⁾ -Arg ^(X6) -Ile-Leu-Gly-Gln ^(X5) -Leu-Tyr (X ²⁾ -

-Ala-Arg (X ⁶⁾ -Lys (X ⁷⁾ -Leu-D-Leu-His (X) -Glu (X ³⁾ -Ile-Met-Asn (X ⁵⁾ -Arg (X ^6), Gln ^(X5 ) -Gin (X ⁵⁾ -Gly-Glu (X ³⁾ -Arg (X ⁶⁾ -Asn (X ⁵⁾ -Gin (X ⁵⁾ -Glu (X ³⁾ -Gin (X ⁵⁾ -Arg (X ⁶ ) -Ser (X ⁴⁾ -Arg ^(X6) -Phe-Asn (X ⁵⁾ -X ⁹ wherein DCB

X ³ means OBzl, X ⁴ means Bzl, X ⁵ means Xan, X ⁶ means Tos, X ⁷ 2C1-Z a X ⁹ is _-O-CH2 -resin padding.

For example, trifluoroacetic acid can be partially or completely removed from the purified fracxane to be used to remove the protecting group from the *-amino group.

The protected peptide is cleaved, stripped and purified in a similar manner to Example 1.

The salts can be removed using a gradient of CH 2 CN in 0.1% trifluoroacetic acid. The middle portion is then lyophilized to give the resulting peptide having a purity of greater than 98%.

The synthesis was repeated using MBHA resin to give the same peptide with an amidated C-terminal end in the manner generally described by Val et al., U.S. Pat. No. 4,292,313 for coupling Asn to MBHA resin.

Example 12

Synthesis of the amide peptide of 40 residues [C ⁰ MeHis @ ^1, D-Leu ^23] -hpGRF (1-40) -NH ₂ having the formula:

MeHis-Ala-Asp-Ala-Ile-Phe-Thr-As-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-D-Leu-Gln -Asp-Ile-Met-Ser-Arg-Gln-Gln-Gly-Glu-Ser-Asn-Gln-Glu-Arg-Gly-Ala-NH ₂ is performed sequentially using a peptide synthesizer (Beckman 990) on MBHA resin as follows as described by Val et al. in U.S. Pat. No. 4,292,313. According to the results of thin layer chromatography and high pressure liquid chromatography, essentially pure peptide is obtained in this way.

Example 13

Synthesis of [N Me-Met ¹ , D-Leu ²³ , Nle ²⁷ ] -rGRF- (1-43) -OH of formula:

Me-Met-Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Ile-Leu-Gly-Gln-Leu-Tyr-Ala-Arg-Lys-Leu-D-Leu-His -Glu-Ile-Nle-Asn-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln-Glu-Gln-Arg-Ser-Arg-Phe-Asn-OH is performed sequentially using a peptide synthesizer (Beckman 990 ) on chloromethylated resin as described in Example 1. According to the results of thin layer and high pressure liquid chromatography, essentially pure peptide was obtained.

Example 14 _v

Synthesis of an analog fragment, i.e. [D-Leu ²³ ] -hpGRF (1-32) -NH _{2 of the} formula:

H-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-As-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln

-Leu-Ser-Ala-Arg-Lys-Leu-D-Leu-Gln-Asp-Ile-Met-Ser-Arg-Gln-Gln-Gly-NH ₂ is performed sequentially using a peptide synthesizer (Beckman 990) on a resin MBHA according to the method described in Example 2. According to the results of thin layer chromatography and high pressure liquid chromatography, this method gives a substantially pure analog.

This process was repeated two more times to give [D-Leu ²³ , D-Glu ²⁵ ] -hpGRF (1-32) -NH 2 and [D-Leu ²³ , Nle ²⁷ ] -hpGRF (1-32) -nh 2.

Example 15

Synthesis of the hpGRF analog fragment, i.

[D-Leu ²³ , D-Asp ²⁵ , Nle ²⁷ ] / hpGRF (1-29) -NH ₂ formulas:

H-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyl-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-D-Leu- Gln-D-Asp-Ile-Nle-Ser-Arg-NH ₂ was carried out sequentially using a peptide synthesizer (Beckman 990) on MBHA resin as described in Example 2. According to the results of thin layer and high pressure liquid chromatography, yields a substantially pure peptide.

Example 16

Synthesis of [D-Leu ²³ , Nle- ²⁷ ] -rGRF (1-29) -NH _{2 of} formula:

H-His-Ala-Asp-Ala-Ile-Phe-Thr-Ser-Tyr-Arg-Arg-Arg-Ile-Leu-Gly-Gln-Leu-Tyr-Ala-Arg-Lys-Leu-D-Leu- His-Glu-Ile-Nle-Asn-Arg-NH ₂ is carried out sequentially using a peptide synthesizer (Beckman 990) on MBHA resin as described in Example 2. Thin layer and high pressure liquid chromatography results in this manner. substantially pure peptide.

The synthesis was repeated to give [D-Leu ^17. Nle ²⁷ ] -rGRF (1-29) -NH ₂ .

Example 17

Synthesis of [D-Glu ²⁵ , Nle ²⁷ ] -hpGRF (1-29) -NH _{2 of} formula:

H-Tyr-Ala-Asp-Ala-Ile-Phe-Thr- Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln- D-Glu-Ile-Nle-Ser-Arg-NH ₂ is carried out sequentially using a peptide synthesizer (Beckman 990) on MBHA resin as described in Example 2. Thin layer chromatography and high pressure chromatography results in this manner. substantially pure peptide.

Example 18

Synthesis of [D-Glu ²⁵ , Nle ²⁷ ] -rGRF (1-29) -NH _{2 of} formula:

H-His-Ala-Asp-Ala-Ile-Phe-Thr-Ser-Tyr-Arg-Arg-Arg-IXe-Leu-GXy-Gln-Leu-Tyr-Ala-Arg-Lys-Leu-Leu-His- D-Glu-Ile-Nle-Asn-Arg-N N was carried out sequentially using a peptide synthesizer (Beckman 990) as described in Example 2. Thin layer chromatography and high pressure chromatography yielded substantially pure peptide.

Example 19

Synthesis of [D-Tyr ¹ , D-Leu ²³ ] pGRF (1-44) -NH _{2 of the} formula:

HD-Tyr-AX-Asp-Ala-lle-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-D-Leu-Gln- Asp-Xle-Met-Ser-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln-Glu-Gln-Gly-Ala-Arg-Val-Arg-LeU-NH ₂ is performed sequentially using a peptide synthesizer ( Beckman 990) on MBHA resin according to the method generally described by Val et al. In U.S. Pat. No. 4,292,313. According to the results of thin layer and high pressure liquid chromatography, essentially pure peptide is obtained.

Example 20

Synthesis of [D-His ^1, D-Leu ^{17 '23,} D-Glu ^25, Nle ^27] -rGRF (1-43) -OH of the formula:

HD-His-Ala-Asp-Ala-Ile-Phe-The-Ser-Ser-Tyr-Arg-Arg-Arg-Ile-Leu-GlyD-Leu-Tyr-Ala-Arg-Lys-Leu-D-Leu-His- D-Glu-Ile-Nle-Asn-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln-Glu-Gln-Arg-Ser-Arg-Phe-Asn-OH is performed (Beckman 990) on chloromethylated resin according to the method described in Example 1. According to the results of thin layer chromatography and high pressure liquid chromatography, this method yields substantially pure peptide.

Example 21

Synthesis of [D-Ala ^2, D-Leu ^{17 '23,} D-Asp ^25] -hpGRF (1-32) -NH ₂ having the formula:

H-Tyr-D-Ala-Asp-Ala-Ile-Phe-Thr- Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-D-Leu-Ser-Ala-Arg-Lys-Leu- _{D-Leu-D-Gln-Asp-Ile-Met-Ser-Arg-Gln-Gln-Gly-NH2} is conducted sequentially with a peptide synthesizer (Beckman 990) on an MBHA resin as described in Example 2. the results chromatography thin layer and high pressure chromatography yielded a substantially pure analog in this way.

Example 22

Synthesis of [D-Tyr ¹ , D-Ala ² , D-Asp ³ , D-Asn ⁸ , D-Tyr ¹⁰ , D-Ala ¹⁵ ,

D-Leu ^{17 '23 25} D-Asp, D-Met ^27] -hpGRF- (1-29) -NH ₂ having the formula:

HD-Tyr-D-Ala-I-Asp-Ala-Ile-Phe-Thr-D-Asn-Ser-D-Tyr-Arg-Lys-ValLeu-D-Ala-Gln-D-Leu-Ser-Ala- Arg-Lys-Leu-D-Leu-Gln-D-Asp-Ile-D-Met-Ser-Arg-NH ₂ was carried out sequentially using a peptide synthesizer (Beckman 990) and MBHA resin as described in Example 2. as a result of thin layer chromatography and high pressure liquid chromatography, a substantially pure analog is obtained.

Example 23

Synthesis of [D-His ¹ , D-Ser ⁸ , D-Leu ²³ , Nle ²⁷ ] -rGRF (1-43) -OH of formula:

HD-His-Ala-Asp-Ala-Ile-Phe-Thr-D-Ser-Ser-Tyr-Arg-Arg-Ile-Leu-Gly-Gln-Leu-Tyr-Ala-Arg-Lys-Leu-D- Leu-His-Glu-Ile-Nle-Asn-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln-Glu-Gln-Arg-Ser-Arg-Phe-Asn-OH is performed sequentially using a peptide synthesizer (Beckman 990) on a chloromethylated resin as described in the example

1. Depending on the result of thin layer chromatography and high pressure liquid chromatography, a substantially pure peptide is obtained in this way.

Example 24

Synthesis rGRF analog fragment, i.e., '[N ^O6 MeTyr ^1, D-Glu ²⁵ -rhGRF (l-29) -NH ₂ having the formula:

N MeTyr - Ala - Asp - Ala - Ile - Phe - Thr - Ser - Ser - Tyr - Arg - Arg - Ile - Leu - Gly - Gln - Leu - Tyr - Ala - Arg - Lys - Leu - Leu - His - D -Glu-Ile-Met-Asn-Arg-nh ₂ is carried out sequentially using a peptide synthesizer (Beckman 990) on MBH resin as described in Example 2. Thin layer chromatography and high pressure liquid chromatography yields essentially pure peptide.

Example 25

Synthesis of [C ⁰⁰ MeLeu ¹ , D-Leu ²³ , D-GLu ²⁵ , Nle ²⁷ ] -rGRF- [Val ⁴⁴ ] -Nh ₂ formulas:

MeLeu-Ala-Asp-Ala-Ile-Phe-Thr-Ser-Tyr-Arg-Arg-Arg-Ile-Leu-Gly-Gln-Leu-Tyr-Ala-Arg-Lys-Leu-D-Leu-His -D-Glu-Il-Nle-Asn-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln-Glu-Gln-Arg-Ser-Arg-Phe-Asn-Val-NH ₂ is carried out sequentially using peptide synthesizer (Beckman 990) on MBHA resin as described in Example 2. Thin layer chromatography and high pressure liquid chromatography yielded substantially pure peptide.

The various synthetic peptides prepared by the method of each example were compared to synthetic hpGRF (1-40) -OH in vitro, and it was shown that they all had no greater ability to promote growth hormone secretion and other similar effects.

In order to determine the efficacy of some synthetic peptides on growth hormone release, in vitro experiments were performed using synthetic hpGRF (1-40) -OH as a standard and equimolar concentrations of various other analogs and fragments. Cultures of rat pituitary cells from glands harvested 3 to 5 days ago were used. Cultures that were optimal for this follow-up were used for comparative assays as described in Vale et al., Endocrinology, 91, 562-572 (1972) and in more detail in Vale et al., Endocrinology (1983) in the press. Incubation with the test substances is carried out for 3 to 4 hours, after which aliquots of the medium are collected and processed by measuring the immunoreactive growth hormone (ir GH) content by a conventional radioimmunoassay using platelets.

The results of this comparison for equimolar concentrations are shown in χ in Table I.

Table I

Peptide comparison in%

hpGRF (1-40) -OH (standard) 100 ALIGN! [D] Glu ²⁵ , Nle ²⁷ ] -rGRF (1-29) -NH ₂ 125 [D-Leu ²³ , Nle ²⁷ ] -rGRF (1-29) -NH ₂ 160 [D-Leu ¹⁷ , Nle ²⁷ ] -rGRF (1-29) -NH ² 138 [N Me Phe ¹ (4Cl)] - hpGRF (1-32) -NH ₂ 12 [C Me Phe ¹ (4Cl)] - hpGRF (1-32) -NH ₂ 4 [401-Phe ¹ ] hpGRF (1-32) -NH ₂ 2

In vitro experiments with these synthetic peptides show that each has the full biological activity of hpGRF (1-40) -OH-. The maximum effective concentration for [D-Leu ²³ , Nle ²⁷ ] -hpGRF (1-29) -NH ₂ is

About 27 nanomoles.

In addition to in vitro growth hormone secretion assays, in vitro experiments were also performed by injecting synthetic peptides into male rats after pretreatment with FLA-63, a dopamine hydroxylose inhibitor that suppressed spontaneous growth hormone secretion without affecting the response to exogenous GRF . Blood samples were taken with a catheter just before injection and 5 and 20 minutes after injection. Growth hormone levels in the blood, measured by radioimmunoassay, show that the synthetic [D-Leu ²³ , Nle ²⁷ ] -rGRF (1,229) -NH 2 other D-Leu ¹⁷ and D-Glu ² -alogues rGRF and the above identified N ^ Me and ⁵⁰ C-Me analogues hpGRF (l-32) -NH2 are powerful stimulators of the secretion of hypophysial growth hormone and much longer duration of action than rGRF (1-29) _-NH2 and hpGRF (l-32) -NH _second Other known GRFs in vivo that are effective in growth hormone secretion have confirmed these results. Doses range from 100 nanograms to 50 micrograms of peptides per kg of weight.

Synthetic analogues of hpGRF and probably also analogues of rGRF and pGRF should be useful in human medicine when it is necessary to increase growth hormone production. Stimulation of growth hormone secretion by these analogues is particularly useful in patients with complete lack or lack of secretion of growth hormone due to the lack of production of this hormone due to the lack of a factor that promotes its release. In addition, it is likely that increased growth hormone secretion and the effect of this secretion on accelerated growth may also favor the development of humans or animals with normal levels of growth hormone. In addition, it is possible in this way to affect the fat cells in the body and alter other metabolic, immunological and developmental processes that depend on the production of growth hormone. Thus, these analogs could be useful for promoting anabolic processes in humans, for example, in severe burns. The administration of these analogues in farm animals such as chickens, turkeys, pigs, goats, cattle and sheep as well as cold-blooded animals such as fish and other marine animals such as turtles or amphibians to accelerate their growth could also be considered. to increase the protein-to-fat ratio, which should at the same time lead to a better utilization of the proteins that are supplied to these animals in the feed.

When administered to humans, the synthetic peptides should have a purity of at least 93%, preferably at least 98%. Purity in this case means the weight percent of the desired peptides in the mixture of other peptides and fragments thereof. When administering synthetic peptides to livestock and other animals to accelerate their growth and reduce fat, a much lower purity, for example only 5%, in some cases a purity of 0.01% is acceptable.

Synthetic peptides or non-toxic salts thereof may be formulated into pharmaceutical compositions in association with conventional pharmaceutically or veterinarily acceptable carriers. The compositions thus obtained may be administered to animals including humans, the usual route of administration being intravenous, subcutaneous, intramuscular, dermal, and optionally intranasal or oral. The administration of the peptides produced by the method of the invention may be decided upon by the patient in need of increased growth hormone release in the interest of the patient. The desired dosage of the peptides may vary according to the conditions, particularly independent of the severity of the patient's condition and the desired duration of treatment.

The peptides produced by the process of the invention can often be administered in the form of their non-toxic salts, for example acid addition salts, or in the form of their complexes with metals, such as zinc, iron, and the like. For the sake of simplicity, these complexes are also considered to be salts. Examples of suitable acid addition salts include hydrochlorides, hydrobromides, sulfates, phosphates, maleates, citrates, acetates, succinate, malate, benzoates, salts of ascorbic acid, tartaric acid and the like. When the active ingredient is to be administered orally in the form of tablets, the tablet may also contain a binder such as tragacanth, corn starch or gelatin, a disintegrating agent such as alginic acid, a glidant such as magnesium stearate and the like. Where administration in liquid form is desired, the composition may contain a sweetener and / or flavoring agent, and intravenous administration will in particular include isotonic sodium chloride solution, phosphate buffer and the like.

The peptides produced by the method of the invention need to be administered to humans under the supervision of a physician, the pharmaceutical compositions will usually contain the appropriate peptide together with a conventional solid or liquid carrier acceptable in a pharmaceutically acceptable manner. For parenteral administration, the active ingredient will generally range from 100 nanograms to 50 micrograms of peptides per kg body weight. "

While the invention has been described in connection with some preferred embodiments, which are currently the best known embodiment, it will be understood that various changes and modifications can be made by those skilled in the art without departing from the scope of the invention. of the invention. For example, various modifications can be made to the backbone peptide chain, in particular to omit some residues or groups of residues from the C-terminal end of the peptide chain in accordance with conventional methods currently used to obtain peptides or peptide fragments that retain all the potency of the parent compound or a substantial portion of the biological activity of the parent peptide. All substances thus obtained are also within the scope of the invention. In addition, residues may be added at or both ends of the peptides, and / or residues may be used in place of naturally occurring residues, which are generally considered to be equivalent to those known in peptide chemistry to produce analogs having at least a substantial portion of the biological activity of the polypeptides without departing from the scope of the invention.

Claims (7)

1. Peptides of general formula R ₁ "R ₂ " R ₃ "Ala" Ile-Phe-ThrR ₈ "Ser-R ₁₀ -Arg ~ R ₁₂ " R ₁₃ "Leu-R ₁₅ Gln-R ₁₇ -R ₁₈ -Ala-Arg-Lys-Leu -R ₂₃ -R ₂ 4-R ₂ 5Ile-R ₂₇ ”R ₂₈ ” Arg-Gln Gln-Gly-GlU “R _{3 4} -Asn“ Gln-GlU “R _{3 8} -R _{3 g} -R _{4 0} Arg ~ R _{4 2} ”R _{4 3} ~ R _{4 4} where R 1 is Tyr, D-Tyr, Met, Phe, D-Phe, pCl "Phe-LeU" His. or D-His, with or without substitution of type C ^alpha Me or N ^{and f and} Me, (I) ^R 2 means Ala or D-Ala, ^R 3 means Asp or D-Asp, ^R 8 means Ser , Asn , D-Ser or ^R 10 means Tyr or D-Tyr, ^R 12 means Arg or Lys, ^R 13 means Ile or Wall, ^R 15 means Gly or D-Ala, ^R 17 means Leu or D-Leu, ^R 18 means Tyr or Ser, ^R 23 means Leu or D-Leu, ^R 24 means His or Gin, ^R 25 means Glu , Asp , D-Glu or ^R 27 means Met , D-Met, Ala, Nle ^R 28 means Asn or Ser ,. ^R 34 means Arg or Ser, ^R 38 means Gin or Arg, ^R 39 means Arg or Gly, ^R 40 means Ser or Ala, ^R 42 means Phe , Ala or Val, R ₄₃ is Asn or Arg, R ₄₄ is Val or Leu, provided that either a) R 1 has a type C ^alpha Me, or N ^alpha Me substitution; or b) R ₁₇ and / or R ₂₃ is D-Leu; c) R ₂₅ is D-Glu or D-Asp, and furthermore it is possible to omit one of the radicals for ^R 44 ^'R 43 ^_R 44' ^R 42 ^'R 43' ^R 44 ' Arg-R ₄₂ -R ₄ gR ₄₄ , ^R 10 ” ^Ar ^“ ^R 42 ^_R 43 ' ^R 44' ^R 39 ^R 40 ^Arg “ ^R 42“ ^R 43 ” ^R 44 · ^R 3 8“ ^R 3 9 ^R 4 0 ^Ar 9 ” ^R 4 2 ^R 4 3” ^R 4 4 ' Glu-Rg ^{R R} 3 9 ** ^R 4 0 ~ ^Ar g ~ R ₄ 2 ^R 4 3 ^R 4 4 ' Gln-Glu-R ₃₈ -R ₃₉ -R ₄₀ -Arg-R ₄₂ -R ₄₃ "R ₄₄ Asn-Gln-Glu-Rg θ ~ ^R gg ” ^R 4 0 ~ arg- ° R _{4 2} “ ' ^R 4 3 * ” ^R 4 4' R ₃₄ 'Asn-Gin-Glu-Rgg-Rg _9- R ₄ q-Arg-R ₄₂ -R ₄ g-R ₄₄ , Glu — Rg ₄ —Asn — Fln — Glu — Rg g “^ 3g” ^R 4 q “Arg — R _{4 2} —R ₄ 3“ R ₄ 4 / Gly-Glu-Rg ₄ -Asn-Gln-Glu-Rg g-Rg ₉ -R ₄₀ -Arg-R _{4 2} -R _{4 3} —R _{4 4} , Gln-Gly-Glu-Rg ₄ -Asn-Gln-Glu-Rgg-Rg g "R4 Q" Arg "R _{4 2} " ^R ₄ g " ^R 44 / Gin — Gin — Gly — Glu — Rg ₄ ”Asn — Gin — Glu — Rg g — Rg ₉ —R ₄ θ —Arg — R _{4 2} —R ₄ g — R ₄ 4 · 2. A process for the production of peptides of the formula I as claimed in claim 1, which comprises: i) binds the N-terminal amino acid protected by the target. or (ii) the protective tert-butoxycarbonyl group is cleaved off and the individual amino acids are sequentially bound in an amount of 1 to 2 mmol of the protected target. butoxycarbonyl group, per gram of resin in methylene chloride, dimethylformamide or a mixture of these solvents to give a compound of formula II with at least one protecting group X ¹ - ^ (X or X ² -R ² -R ^{3 -} (X ³ ) -Ala-Ile-Phe-Thr (X ⁴ ) -R 8 (X ⁴ or X ⁵ ) -Ser (X ⁴ ) -R 10 (X ² ) "Arg (X ⁶⁾ -R 12 (X ⁶ or X ⁷⁾ -R 13 -R ₁₅ Leu Gln (X ⁵⁾ -R17R18 (X ²⁾ Ala-Arg (X ⁶⁾ -Lys (X ⁷⁾ -Leu _R23-R24 (X or X ⁵⁾ -R 25 (X ³⁾ -Ile-R 27 -R ₂₈ (X ⁴ X ^{5 neb0)} -Arg (X ⁶⁾ 'Gln (X ⁵⁾ -Gln »(X5) -Gly -Glu ^(X3) -R34 (X ⁴ or X ⁶⁾ -Asn (X ⁵⁾ -Gln (X ⁵⁾ -Glu (X ³⁾ R3g (X ⁵ or X ⁶⁾ -R 39 (X ⁶⁾ -R 40 ( X ⁴ ) -Arg (X ⁶ ) -R ₄₂ -R ₄₃ (X ⁵ or X ⁶ ) -R ₄₄ -NH-resin, (II) wherein X ^J represents a hydrogen atom or an amino group, a protecting group on alpha-X 'represents a hydrogen atom on a hydrophenyl group Tyr, or a protecting group on a phenol group X 'represents a hydrogen atom or an Asp or Glu protecting group, a carboxy group X' X 'represents a hydrogen atom or a protecting group on an alcohol hydro group Thr, represents a hydrogen atom or a protecting group on an alcoholic hydroxy group Ser, represents a hydrogen atom or a protecting group on a guanidine group Arg, represents a hydrogen atom or a protecting group on an amino group Lys iii) cleaving the protecting group or protecting groups from the compound of formula II to form the peptides of formula I or a biologically active fragment thereof, and optionally iv) converting the resulting peptide into its non-toxic addition salt. 3. The method according to claim 2, characterized in that by locally _R25 bound D-Glu. 4. Method according to claim 2, characterized in that locally binds R ₂₅ is D-Asp. 5. A method according to claim 2, characterized in that instead of R ₂₃ bound D-Leu. 6. Method according to points 2 to 5, characterized se by that se on spot R _1? D-Leu. 7. Method according to points 2 to 6, characterized se by that se on spot R ₂ 7 bind Nle. 8. Method according to points 2 to 7, characterized se by that se on spot R ^{1 is} attached ^{and the} C @ ^.alpha Me or N ^{all 'and} Me. 9. A process according to claim 7, wherein the protecting group X ' butoxycarbonyl such as X ² is introduced 2,6-dichlorobenzyl, as X ³ is introduced benzyl, as X ⁶ is introduced tosyl and as X ^{7 is} 2-chlorobenzyloxycarbonyl. 10. A pharmaceutical composition for stimulating the release of growth hormone in an animal comprising, as an active ingredient, a peptide of formula (I) or a non-toxic salt thereof and a pharmaceutically acceptable carrier.