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GB2118190A - Peptides with sauvagine-like activity - Google Patents

Peptides with sauvagine-like activity Download PDF

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Publication number
GB2118190A
GB2118190A GB08309547A GB8309547A GB2118190A GB 2118190 A GB2118190 A GB 2118190A GB 08309547 A GB08309547 A GB 08309547A GB 8309547 A GB8309547 A GB 8309547A GB 2118190 A GB2118190 A GB 2118190A
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leu
ser
pro
asp
lle
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GB8309547D0 (en
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Castiglione Roberto De
Giuseppe Perseo
Franesco Santangelo
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Pfizer Italia SRL
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Farmitalia Carlo Erba SRL
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575Hormones
    • C07K14/57509Corticotropin releasing factor [CRF] (Urotensin)
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02PCLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00Technologies relating to chemical industry
    • Y02P20/50Improvements relating to the production of bulk chemicals
    • Y02P20/55Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

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  • Toxicology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Endocrinology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

There are provided peptides of the formula X-A-Pro-Pro-Ile-Ser-B-C-Leu-D-E-F-G-W wherein X is H, a terminal N atom protecting group, or a residue of a natural L-amino acid, optionally protected by a terminal N atom protecting group; A is a L-amino acid residue or a glycine residue; B is a neutral L-amino acid residue; C is a neutral or acidic L-amino acid residue; D is a L-amino acid residue which contains in the side chain an alcoholic hydroxy group, optionally protected by a conventional hydroxy protecting group; E is a valence bond or a neutral L-amino acid residue; F is a valence bond or a L-amino acid residue; G is a valence bond or a neutral L-amino acid residue; W is OH, NH2, OR, NHR, NR2 or NH-NH-R'; wherein R and R' are optionally substituted hydrocarbon. Pharmaceutically acceptable salts of these peptides are also provided.

Description

SPECIFICATION Biologically active peptides The invention relates to biologically active peptides, their pharmaceutically acceptable salts, processes for their preparation and pharmaceutical compositions containing them.
The peptides of the invention are fragments or modified fragments of sauvagine, a natural peptide consisting of 40 amino acid residues (P. C. Montecucchi, A. Henschen -- Int. J. Peptide Protein Res. 18, 113-120, 1981), which shows a series of interesting pharmacological activities, such as release of ACTH and p-endophin, and inhibition of the release of growth hormone, TSH and prolactin (P. C.
Montecucchi et al., "Hormone Receptor in Digestion and Nutrition", G. Rosselin et al. eds., Elsevier/North-Holiand Biomed. Press, 1979,101-108).
It was surprisingly found that the peptides of the invention maintain totally or partially the biological activity spectrum of sauvagine. The importance of this discovery is evident. The synthesis of a tridecapeptide or a shorter homologue is, obviously, more easily realized than the synthesis of a peptide of 40 amino acid residues. Purer or more easily purifiable products are obtained.
In this specification symbols and abbreviations are those commonly used in peptide chemistry (see J. Biol. Chem., 1 972, 247, 977-983).
The invention provides peptides of the general formula: X-A-Pro-Pro-lle-Ser-B-C-Leu-D-E-F-G-W wherein: X represents a hydrogen atom, a terminal nitrogen protecting group of acyl, aromatic urethane, alkyl, aralkyl or aliphatic urethane type or a residue of a neutral L-amino acid, the free amino group of the residue optionally being protected by a terminal nitrogen atom protecting group of the type cited above; A represents a L-amino acid residue or a glycine residue; B represents a neutral L-amino acid residue; C represents a neutral L-amino acid residue or an acidic L-amino acid residue; D represents a L-amino acid residue which contains in the side chain an alcoholic hydroxy group, which may be free or protected by a conventional hydroxy protecting group; E represents a valance bond or a neutral L-amino acid residue;; F represents a valence bond or a L-amino acid residue: G represents a valence bond or a neutral L-amino acid residue; W represents a hydroxy group, an amino group'or a group of the formula OR, NHR, NR2 or NH-NH-R1 wherein R represents a straight chain, branched chain or cyclic (including fused or bridged ring) alkyl group having up to 11 carbon atoms, and being substituted or unsubstituted, a phenyl group or an aralkyl group having from 6 to 8 carbon atoms; and R' represents a hydrogen atom, any of the groups which R may represent, an alkenyl group having 3 to 8 carbon atoms; a straight chain, branched chain or cyclic aliphatic acyl group having from 1 to 11 carbon atoms, unsubstituted or substituted by a hydroxy or amino group or a halogen atom, an aromatic acyl group, unsubstituted or substituted by a hydroxy or amino group or a halogen atom, a straight chain, branched chain or cyclic aliphatic urethane type group having from 3 to 11 carbon atoms, or an aromatic urethane type group.
Preferred terminal nitrogen atom protecting groups which X may represent include (of acyl type) formyl, acetyl, trifluoroacetyl, propionyl and benzoyl groups; (or aromatic urethane type) benzyloxycarbonyl (Z), 4-nitrobenzyloxycarbonyl, 4-methoxybenzyloxyca rbonyl, 2,4- dichlorobenzyloxycarbonyl, 2-bromobenzyloxycarbonyl, 94luornnylmethoxycarbonyl (Fmoc) and 3,5 dimethoxy-a,a'-dimethylbenzyloxycarbonyl (Ddz) groups; (of aliphatic urethane type) t-butoxycarbonyl (Boc), 1 -methyl-cyclobutoxycarbonyl, adamantyloxycarbonyl and isobornyloxycarbonyl groups; and (of alkyl and aralkyl type) trityl, benzyl (Bzl), methyl and isopropyl groups. Preferred neutral L-amino acid resides which X may represent include Pyr, Gln, Pro and 2-oxo-L-pipecolic acid.Preferred L-amino acid residues which A may represent include Glu and Gln. Preferred neutral L-amino acid residues which B may represent include lle, Leu, Nle, Val and Phe. Preferred neutral L-amino acid residues which C may represent include Asn and Gln; preferred acidic L-amino acid residues which C may represent include Asp and Glu. Preferred L-amino acid residues which D may represent include Ser, Hse, and Thr; if the hydroxy group of a residue represented by D is protected, it is preferably protected by a t-butyl, trityl, benzyl, 2,4-dichlorobenzyl, benzyloxycarbonyl, 2-bromobenzyloxycarbonyl, tetrahydropyranyl or tbutoxycarbonyl group or by a lower acyl group such as a formyl, acetyl, trifluoroacetyl, propionyl or benzoyl group.
Preferred neutral L-amino acid residues which E may represent include Phe, Leu and Nle. Preferred L-amino acid residues which F may represent include Glu, Gln and His. Preferred neutral L-amino acid residues which G may represent include Leu and Phe.
Preferred groups which R may represent include methyl, ethyl, n-propyl, isopropyl, n-butyl, s-butyl, isobutyl, t-butyl, 2,2,2-trifluoroethyl, cyclohexyl, adamantyl, phenyl, benzyl and phenethyl groups.
Examples of alkenyl groups which R' may represent include allyl and pentenyl groups. Examples of acyl groups which R' may represent are formyl, acetyl, trifluoroacetyl, propionyl, butyryl, adamantylcarbonyl, benzoyl, phenylacetyl and cinnamyl groups. The aliphatic and aromatic urethane type groups which R' may represent are preferably those groups mentioned as preferred terminal nitrogen protecting groups X of aliphatic and aromatic urethane type.
Salts of peptides according to the invention with pharmaceutically acceptable acids or bases are within the scope of the invention. Such acid addition salts can be derived from a variety of inorganic and organic acids such as sulphuric, phosphoric, hydrochloric, hydrobromic, hydroiodic, nitric, sulphamic, citric, iactic, pyruvic, oxalic, maleic, succinic, tartaric, cinnamic, acetic, trifluoroacetic, benzoic, salicyclic, gluconic, ascorbic and related acids. Such base addition salts can be derived from a variety of inorganic and organic bases as sodium hydroxide, potassium hydroxide, diethylamine, triethylamine and dicyclohexylamine.
The synthesis of the peptides of the invention may be accomplished either by classical solution methods or by solid phase on polymeric supports. In the classical solution method the synthesis consists essentially in appropriate successive condensations of protected amino acids or peptides. The condensation is carried out so that the resulting peptides have the desired sequence of 9 to 13 amino acid residues. The amino acids and peptides, which are condensed according to methods known in themselves in polypeptide chemistry, have such of their amino and carboxy groups as are not involved in the formation of the peptide linkage blocked by a suitable protecting group. The hydroxy functions of hydroxy amino acids may be protected by suitable protecting groups (throughout all the synthesis or only during a few steps) or may be kept unprotected.
The protecting groups are capable of being removed by acidolysis, saponification or hydrogenolysis. For the protection of the amino groups the following protective groups may for example be employed; benzyloxycarbonyl, t-butoxycarbonyl, trityl, formyl, trifluoroacetyl, o- nitrophenylsulphenyl, 4-methoxybenzyloxycarbonyl, 9-fluorenylmethoxycarbonyl or 3,5-dimethoxy u.u'-dimethylbenzyloxycarbonyl. For the protection of the carboxy groups the following protective groups may for example be employed: methyl, ethyl, t-butyl, benzyl or p-nitro-benzyl. For the protection of the hydroxy groups the following protecting groups may for example be used: acetyl, tbutoxycarbonyl, benzyloxycarbonyl, 2-bromobenzyloxycarbonyl, tetrahydropyranyl, t-butyl, trityl, benzyl or 2,4-dichlorobenzyl.
The condensation between an amino group of one molecule and a carboxyl group of another molecule to form the peptide linkage may be carried out through an activated acyl-derivative such as a mixed anhydride, an azide or an activated ester, or by direct condensation between a free amino group and a free carboxyl group, in the presence of-a condensing agent such as dicyclohexylcarbodiimide, alone or together with a racemization preventing agent, such as N-hydroxysuccinimide or 1 hydroxybenzotriazole.
Hydrazido or substituted hydrazido derivatives according to the invention are prepared by condensation of the.N-protected peptide or amino acid with a suitably substituted hydrazido such as benzylcarbazate, t-butylcarbazate, adamantylcarbazate, phenylhydrazine or adamantylhydrazine, or reacting the N-protected peptide or amino acid hydrazide with a suitable alkylating agent, such as an alkyl chloride, or with a suitable acylating agent such as benzylchloroformate, t-butylchloroformate, di-t butyldicarbqnate or adamantylfluoroformate.
The condensation may be carried out in a solvent such as dimethylformamide, pyridine, acetonitrile, tetrahydrofuran or N-methyl-2-pyrrolidone. The reaction temperature may be from -300C to ambient temperature. The reaction time is generally from 1 to 120 hours. The scheme of synthesis, the protecting groups and the condensing agents are selected so as to avoid the risk of racemization.
D-protecting reactions are carried out according to methods known per se in polypeptide chemistry. Peptides wherein W represents OR are prepared, for example, starting from the C-terminal amino acid esterified by the appropriate alcohoi. Peptides wherein W represents OH can be prepared, for example, by hydrolysis of peptides wherein W represents OR. Peptides wherein W represents NH2, NHR or NR2 can be prepared by ammonolysis of the corresponding esters or starting from a C-terminal amino acid amidated by an appropriate amine.
In the solid-phase method a polymeric support is used. The polymer is preferably a copolymer of styrene with from 1 to 2 percent by weight of divinyl benzene as a cross-linking agent which causes the polystyrene polymer to be completely insoluble in most organic solvents. The synthesis is commenced from the C-terminal end of the peptide, by attaching the required amino acid to a chloromethylated resin, a hydroxymethyl resin, or a benzhydrylamine resin. The amino and side chain protecting groups are those described in the classical solution synthesis.
In the preparation of the compounds of this invention, an amino protected amino acid is coupled to the chloromethylated resin via caesium salt, or to a hydroxymethyl or benzhydrylamine resin, with the aid of a condensing agent such as dicyclohexylcarbodiimide.
After the initial coupling, the amino protecting group is removed by a choice of reagents including trifluoroacetic acid or hydrogen chloride solutions in organic solvents at room temperature. After removal of the amino protecting group, the remaining protected amino acids are coupled stepwise in the desired order to'obtain the desired peptide. Each protected amino acid is generally reacted in)a 3fold excess using an appropriate carboxy group activator such as dicyclohexylcarbodiimide in solution in, for example, methylene dichloride: dimethylformamide mixtures.
After the desired amino acid sequence has been completed, the peptide is removed from the resin support by treatment with a reagent such as hydrogen fluoride, which not only cleaves the peptides from the resin, but also cleaves most of the remaining side-chain protecting groups. When chloromethylated or hydroxymethylated resin is used, hydrogen fluoride treatment results in the formation of the free peptide acid (W = OH). When benzhydrylamine resin is used, hydrogen fluoride treatment results directly in the free peptide amide (W = NH2). Alternatively, when the chloromethylated or hydroxymethylated resin is employed, the side-chain protected peptide can be cleaved by treatment of the peptide resin with ammonia or a mono- or di-alkylamine to give the desired side-chain protected amide, alkylamide or dialkylamide (W = NH2, NHR, NR2).Side-chain protection may then be removed by any of the methods known in the art. In preparing the esters of the present invention (W = OR), the resins used to prepare the acid (W = OH) are employed and the side-chain protected peptide is cleaved with a base and the appropriate alcohol. Side-chain protection is then removed in the usual way.
Alternatively, the peptide acids and amides can be obtained from the peptide esters by saponification or ammonolysis.
The compounds according to the invention show an interesting pharmacological activity on inhibition of prolactin release. In addition some of the compounds of the invention are endowed with the following pharmacological activities: release of ACTH and p-endorphin, inhibition of the release of growth hormone and TSH. The inhibition of prolactin secretion was determined both "in vitro" and "in vivo"; "in vitro" test by using pituitary celis (obtained from rat or beef) suspended in Bio-gel columns, "in vivo" test by measuring the inhibition of serum basal prolactin secretion in male rats and the inhibition of suckening stimulated prolactin secretion in lactating rats.
The inhibition of prolactin release was determined in vitro using the method described by P. J.
Lowry in J. Endocrinol. 63, 1 63 (1974). The peptides synthesized were able to inhibit by 6080% prolactin secretion in isolated and perfused rat pituitary cells at a concentration ranging from 10-4 to 1O0M.
The compounds according to the invention are endowed with anxiolytic activity determined by modifications of the qualitative and quantitative spontaneous activity in rats according to the method described by A. E. Whimbey and V. -H. Denenberg in J. Comparative Physiology and Psychology, 63, 500-504 (1967).
The synthesized compounds, administered subcutaneously to rat at doses ranging from 20 mcg to 100 mcg/kg were-able to decrease the level of anxiety induced by a new environment (open-field), and to increase explorativity in familiar environments (residential cages).
The invention further provides a pharmaceutical composition comprising a compound of the present invention or.a pharmaceutically acceptable salt thereof in admixture with a pharmaceutically acceptable diluent or carrier; in addition, these preparations can have direct or delayed liberation of the active ingredient.
The preferred peptides according to the invention are the following: 1. H-Pyr-Gly-Prn-P ro-Ile-Ser- le-Asp-Leu-Ser-Leu-Gl u-Leu-OH 2. H-Pyr-G Iy-Pro-P ro-l le-Ser-lie-Asp-Leu-Ser-Leu-G lu-Leu-N Hz 3. H-Pyr-Gly-P ro-P ro-lle-Ser-lle-Asp-Leu-Ser-Leu-G lu-Phe-OH 4. H-Pyr-Gly-Pro-Pro-lle-Ser-lle-Asp-Leu-Ser-Leu-Glu-Phe-NH2 5. H-Pyr-G Iy-P ro-P ro-lle-Ser-l le-Asp-Leu-Ser-Leu-G In-Leu-OH 6. H-Pyr-Gly-Pro-Pro-lle-Ser-lle-Asp-Leu-Ser-Leu-Gln-Leu-NH2 7. H-Pyr-Gly-P ro-Pro-lle-Ser-l le-Asp-Leu-Ser-Leu-His-Leu-OH 8.H-Pyr-Gly-Pro-Pro-lle-Ser-lle-Asp-Leu-Ser-Leu-His-Leu-N H2 9. H-Pyr-Gly-Pro-Pro-lle-Ser-l le-Asp-Leu-Ser-Phe-Glu-Leu-OH 10. H-Pyr-G ly-P ro-Pro-lle-Ser-lle-Asp-Leu-Ser-Phe-Glu-Leu-NH2 11. H-Pyr-Gly-Pro-Pro-lle-Ser-lle-Asp-Leu-Thr-Leu-Glu-Leu-OH 1 2. H-Pyr-Gly-Pro-Pro-lle-Ser-lle-Asp-Leu-Thr-Leu-Glu-Leu-NH2 13. H-Pyr-Gly-P ro-P ro-lle-Ser-l le-Asn-Leu-Ser-Leu-Glu-Leu-OH 14. H-Pyr-Gly-Pro-P ro-lle-Ser-l le-Asn-Leu-Ser-Leu-G lu-Leu-N H2 1 5.H-Pyr-Gly-Pro-P ro-l le-Ser-l le-Glu-Leu-Ser-Leu-G lu-Leu-OH 1 6. H-Pyr-Gly-Pro-P ro-l le-Ser-lle-G lu-Leu-Ser-Leu-G lu-Leu-NH2 17. H-Pyr-Gly-Pro-Pro-l le-Ser-Leu-Asp-Leu-Ser-Leu-G lu-Leu-OH 1 8. H-Pyr-Gly-Pro-Pro-lle-Ser-Leu-Asp-Leu-Ser-Leu-Glu-Leu-NH2 19. H-Pyr-Gly-P ro-Pro-lle-Ser-P he-Asp-Leu-Ser-Leu-Glu-Leu-OH 20. H-Pyr-Gly-P ro-Pro-lle-Ser-P he-Asp-Leu-Ser-Leu-Glu-Leu-N H2 21. H-Pyr-Glu-Pro-Pro-lle-Ser-lle-Asp-Leu-Ser-Leu-Glu-Leu-OH 22.H-Pyr-Glu-P ro-P ro-lle-Ser-l le-Asp-Leu-Ser-Leu-Glu-Leu-NH2 23. H-Pyr--Gln-Prn-Prn-Ile-Ser-lIe-Asp-Leu-Ser-Leu-Glu-Leu-OH 24. H-Pyr-Gln-Pro-Pro-lle-Ser-lle-Asp-Leu-Ser-Leu-Glu-Leu-NH2 25. H-Gln-Gly Pro-Pro-lle-Ser-lle-Asp-Leu-Ser-Leu-Glu-Leu-OH 26. H-Gln-Gly-Pro-P ro-lie-Ser-l le-Asp-Leu-Ser-Leu-Glu-Leu-NH2 27. H-Pyr-Gly-P ro-Pro-lle-Ser-l le-Asp-Leu-Ser-P he-His-Leu-OH 28. H-Pyr-G ly-P ro-P ro-l le-Ser-lle-Asp-Leu-Ser-P he-His-Leu-NH2 29. H-Pyr-G ly-Pro-P ro-lle-Ser-lle-Asp-Leu-Thr-P he-His-Leu-OH 30. H-Pyr-Gly-Pro-Pro-Ile-Ser-l Ie-Asp-Leu-Thr-P he-His-Leu-N H2 31. H-Pyr-Gly-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-OH 32.H-Pyr-Gly-Pro-P ro-l le-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-NH2 33. H-Pyr-Glu-Pro-Pro-l le-Ser-Leu-Asp-Leu-Thr-P he-His-Leu-OH 34. H-Pyr-Glu-P ro-P ro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-NH2 35. H-Gln-Glu-Prn-Prn-Ile-Ser-Leu-Asp-LeuJhr-Phe-His-Leu-OH 36. H-Gln-Glu-Prn-Prn-Ile-Ser-Leu-Asp-LeuJhr-Phe-His-Leu-NH2 37. H-Gln-Glu-Pro-Pro-lle-Ser-lle-Asp-Leu-Ser-Leu-Glu-Leu-OH 38. H-Gln-Glu-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser-Leu-Glu-Leu-NH2 39. H-Gln-GIu-P rn-P ro-IIe-Ser-Leu-Asp-Leu-Ser-Leu-Glu-Leu-OH 40. H-Gln-Glu-Pro-Pro-lle-Ser-Leu-Asp-Leu-Ser-Leu-Glu-Leu-NH 41. H-Gln-Glu-P ro-Pro- le-Ser-Leu-Asp-Leu-Thr-Leu-Glu-Leu-OH 42.H-Gln-G lu-P ro-P ro-l le-Ser-Leu-Asp-Leu-Thr-Leu-Glu-Leu-NH2 43. H-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-Glu-Leu-OH 44. H-Gln-Glu-P ro-P ro-lle-Ser-Leu-Asp-Leu-Thr-Phe-G lu-Leu-NH 45. H-Pyr-Gly-Pro-Pro-lle-Ser-lle-Asp-Leu-Ser-Leu-Glu-OH 46. H-Pyr-Gly-P ro-P ro-lle-Ser-lle-Asp-Leu-Ser-Leu-GI u-NH2 47. H-Pyr-Gly-Pro-P ro-lle-Ser-l le-Asp-Leu-Ser-Leu-His-OH 48. H-Pyr-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser-Leu-His-NH2 49. H-Pyr-G ly-P ro-P ro-l le-Ser-lle-Asp-Leu-Ser-P he-Glu-OH 50. H-Pyr-Gly-Pro-Pro-lle-Ser-lle-Asp-Leu-Ser-Phe-Giu-NH2 51. H-Pyr-Gly-Pro-P ro-lle-Ser-lle-Asp-Leu-Thr-Leu-Glu-OH 52. H-Pyr-Gly-Pro-Pro-lle-Ser-lle-Asp-Leu-Thr-Leu-Glu-NH2 53.H-Pyr-Gly-P ro-P ro-lle-Ser-l le-Asn-Leu-Ser-Leu-Glu-OH 54. H-Pyr-Gly-Pro-Pro-lle-Ser-lle-Asn-Leu-Ser-Leu-Glu-NH2 55. H-Pyr-Gly-Pro-Pro-lle-Ser-lle-GI u-Leu-Ser-Leu-Glu-OH 56. H-Pyr-Gly-P ro-P ro-lle-Ser-lle-G lu-Leu-Ser-Leu-Glu-NH2 57. H-Pyr-Gly-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Ser-Leu-Glu-OH 58. H-Pyr-Gly-Pro-Pro-lle-Ser-Leu-Asp-Leu-Ser-Leu-Glu-NH2 59. H-Pyr-Gly-Pro-P ro-l le-Ser-P he-Asp-Leu-Ser-Leu-G lu-OH 60. H-Pyr-Gly-Pro-Pro-lle-Ser-P.he-Asp-Leu-Ser-Leu-Glu-NH2 61. H-Pyr-Glu-Pro-Pro-lie-Ser Ile-Asp-Leu-Ser-Leu-Glu-OH 62. H-Pyr-Gíu-Pro-Pro-lle-Ser-lle-Asp-Leu-Ser-Leu-Glu-NH2 63. H-Pyr-Gln-Pro-Pro-lle-Ser-lle-Asp-Leu-Ser-Leu-Glu-OH 64.H-Pyr-Gln-P ro-P ro-lle-Ser-l le-Asp-Leu-Ser-Leu-Glu-NH2 65. H-Gln-Gly-Pro-Pro-lle-Ser-lle-Asp-Leu-Ser-Ley-Glu-OH 66. H-Gln-G Iy-P ro-P ro-l le-Ser-lle-Asp-Leu-Ser-Leu-Glu-NH2.
67. H-Gln-Glu-Pro-P ro-lle-Ser-Leu-Asp-Leu-Thr-P he-His-OH 68. H-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-NH2 69. H-Pyr-G Iy-P ro-P ro-lle-Ser-lle-Asp-Leu-Ser-Leu-OH 70. H-Pyr-Gly-P ro-P ro-l le-Ser-lle-Asp-Leu-Ser-Leu-NH2 71. H-Pyr-Gly-P ro-P ro-l le-Ser-l le-Asp-Leu-Ser-Phe-OH 72. H-Pyr-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser-Phe-NH2 73. H-Pyr-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Thr-Leu-OH 74. H-Pyr-Gly-Pro-Pro-lle-Ser-lle-Asp-Leu-Thr-Leu-NH2 75. H-Pyr-Gly-Pro-Pro-Ile-Ser-Ile-Asn-Leu-Ser-Leu-OH 76. H-Pyr-Gly-P ro-P ro-lle-Ser-lle-Asn-Leu-Ser-Leu-NH2 77. H-Pyr-Gly-Pro-Pro-lle-Ser-lle-Glu-Leu-Ser-Leu-OH 78. H-Pyr-Gly-Pro-Pro-lle-Ser-lle-Glu-Leu-Ser-Leu-NH2 79. H-Pyr-Gly-Pro-Pro-lle-Ser-Leu-Asp-Leu-Ser-Leu-OH 80. H-Pyr-Gly-P ro-P ro-ile-Ser-Leu-Asp-Leu-Ser-Leu-N H2 81. H-Pyr-G Iy-P ro-P ro-l le-Ser-P he-Asp-Leu-Ser-Leu-OH 82. H-Pyr-G Iy-P ro-P ro-lle-Ser-P he-Asp-Leu-Ser-Leu-NH2 83. H-Pyr-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser-Leu-OH 84. H-Pyr-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser-Leu-NH2 85. H-Pyr-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser-Leu-OH 86. H-Pyr-Gln-Pro-P ro-lle-Ser-l le-Asp-Leu-Ser-Leu-NH2 87; H-Gln-Gly-Pro-Pro-lle-Ser-lle-Asp-Leu-Ser-Leu-OH 88. H-Gln-G Iy-P ro-P ro-lle-Ser-l le-Asp-Leu-Ser-Leu-NH2 89. H-Pyr-Gly-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-OH 90. H-Pyr-Gly-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-NH2 91. H-Pyr-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser-OH 92. H-Pyr-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser-NH2 93. H-Pyr-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Thr-OH 94.H-Pyr-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Thr-NH2 95. H-Pyr-Gly-Pro-Pro-Ile-Ser-Ile-Asn-Leu-Ser-OH 96. H-Pyr-Giy-Pro-P ro-l le-Ser-lle-Asn-Leu-Ser-NH2 97. H-Pyr-Gly-Pro-Pro-Ile-Ser-Ile-Glu-Leu-Ser-OH 98. H-Pyr-Gly-Pro-Pro-Ile-Ser-Ile-Glu-Leu-Ser-NH2 99. H-Pyr-Gly-Pro-P ro-lle-Ser-Leu-Asp-Leu-Ser-OH 100. H-Pyr-Gly-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Ser-NH2 101. H-Pyr-Gly-Pro-Pro-Ile-Ser-Phe-Asp-Leu-Ser-OH 102. H-Pyr-Gly-Pro-Pro-Ile-Ser-Phe-Asp-Leu-Ser-NH2 103. H-Pyr-Glu-Pro-Pro-lle-Ser-lle-Asp-Leu-Ser-OH 104. H-Pyr-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser-NH2 105. H-Pyr-Gln-Pro-Pro-lle-Ser-lle-Asp-Leu-Ser-OH 106. H-Pyr-Gln-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Ser-NH2 107. H-Gln-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser-OH 108. H-Gln-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser-NH2 109.H-Gln-Glu-Pro-Pro-Ile-Ser-Ile-Leu-Asp-Leu-Thr-OH 110. H-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Leu-Asp-Leu-Thr-NH2 111. H-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser-OH 112. H-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser-NH2 113. H-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Thr-OH 114. H-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Thr-NH2 115. H-Gly-Pro-Pro-Ile-Ser-Ile-Asn-Leu-Ser-OH 116. H-Gly-Pro-Pro-Ile-Ser-Ile-Asn-Leu-Ser-NH2 117. H-Gly-Pro-Pro-Ile-Ser-Ile-Glu-Leu-Ser-OH 118. H-Gly-Pro-Pro-Ile-Ser-Ile-Glu-Leu-Ser-NH2 119. H-Gly-Pro-P ro-lle-Ser-Leu-Asp-Leu-Ser-OH 120. H-Gly-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Ser-NH2 121. H-Gly-Pro-Pro-Ile-Ser-Phe-Asp-Leu-Ser-OH 122. H-Gly-Pro-Pro-Ile-Ser-Phe-Asp-Leu-Ser-NH2 123. H-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser-OH 124. H-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser-NH2 125. H-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-OH 126. H-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-NH2 127.H-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser-Leu-OH 128. H-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser-Leu-NH2 129. H-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser-Phe-OH 139. H-Gly-Pro-Pro-lle-Ser-lle-Asp-Leu-Ser-Phe-NH2 131. H-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Thr-Leu-OH 132. H-Gly-Pro-Pro-lle-Ser-lie-Asp-Leu-Thr-Leu-NH2 133. H-Gly-Pro-Pro-Ile-Ser-Ile-Asn-Leu-Ser-Leu-OH 134. H-Gly-Pro-Pro-Ile-Ser-Ile-Asn-Leu-Ser-Leu-NH2 135. H-Gly-Pro-Pro-Ile-Ser-Ile-Glu-Leu-Ser-Leu-OH 136.H-Gly-Pro-Pro-Ile-Ser-Ile-Glu-Leu-Ser-Leu-NH2 137. H-Gly-Pro-Pro-lle-Ser-Leu-Asp-Leu-Ser-Leu-OH 138. H-Gly-Pro-Pro-lle-Ser-Leu-Asp-Leu-Ser-Leu-NH2 139. H-Gly-Pro-Pro-lle-Ser-Phe-Asp-Leu-Ser-Leu-OH 140. H-Gly-Pro-Pro-lle-Ser-Phe-Asp-Leu-Ser-Leu-NH2 141. H-Glu-Pro-Pro-lle-Ser-lle-Asp-Leu-Ser-Leu-OH 142. H-Glu-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser-Leu-NH2 143. H-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-Oh 144. H-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-NH2 145. H-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser-Leu-Glu-OH 146. H-Gly-Pro-Pro-lle-Ser-lle-Asp-Leu-Ser-Leu-Glu-NH2 147. H-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser-Leu-His-OH 148. H-Gly-Pro-Pro-lle-Ser-lle-Asp-Leu-Ser-Leu-His-NH2 149. H-Gly-Pro-Pro-lle-Ser-lle-Asp-Leu-Ser-Phe-Glu-OH 150.H-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser-Phe-Glu-NH2 151. H-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Thr-Leu-Glu-OH 152. H-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Thr-Leu-Glu-NH2 1 53. H-Gly-Pro-Pro-lle-Ser-lle-Asn-Leu-Ser-Leu-Glu-OH 154. H-Gly-Pro-Pro-Ile-Ser-Ile-Asn-Leu-Ser-Leu-Glu-NH2 155. H-Gly-Pro-Pro-Ile-Ser-Ile-Glu-Leu-Ser-Leu-Glu-OH 156. H-Gly-Pro-Pro-Ile-Ser-Ile-Glu-Leu-Ser-Leu-Glu-NH2 157. H-Gly-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Ser-Leu-Glu-OH 1 58. H-Gly-P ro-Pro-i le-Ser-Leu-Asp-Leu-Ser-Leu-Glu-NH2 159. H-Gly-Pro-Pro-Ile-Ser-Phe-Asp-Leu-Ser-Leu-Glu-OH 160. H-Gly-Pro-Pro-Ile-Ser-Phe-Asp-Leu-Ser-Leu-Glu-NH2 161. H-Glu-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser-Leu-Glu-OH 1 62. H-Glu-Pro-Pro-lle-Ser-lle-Asp-Leu-Ser-Leu-Glu-NH2 163.H-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-OH 1 64. H-Glu-Pro-P ro-l le-Ser-Leu-Asp-Leu-Thr-Phe-His-NH2 165. H-Gly-P ro-P ro-lle-Ser-l le-Asp-Leu-Ser-Leu-Glu-Leu-OH 166. H-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser-Leu-Glu-Leu-NH2 167. H-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser-Leu-Glu-Phe-OH 1 68. H-Gly-Pro-Pro-lle-Ser-lle-Asp-Leu-Ser-Leu-Glu-Phe-N2 169. H-Gly-P ro-P ro-l le-Ser-Ile-Asp-Leu-Ser-Leu-His-Leu-OH 170. H-Gly-P ro-P ro-l le-Ser-l le-Asp-Leu-Ser-Leu-His-Leu-NH2 171.H-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser-Phe-Glu-Leu-OH 172. H-Gly-Pro-Pro-l le-Ser-l le-Asp-Leu-Ser-Phe-Glu-Leu-NH2 173. H-Gly-Pro-Pro-lle-Ser-lle-Asp-Leu-Thr-Leu-Glu-Leu-OH 1 74. H-Gly-P ro-P ro-l le-Ser-lle-Asp-Leu-Thr-Leu-Giu-Leu-NH2 1 75. H-Gly-P ro-P ro-lle-Ser-l le-Asn-Leu-Ser-Leu-Glu-Leu-OH 176. H-Gly-Pro-Pro-Ile-Ser-Ile-Asn-Leu-Ser-Leu-Glu-Leu-NH2 177. H-Gly-Pro-Pro-Ile-Ser-Ile-Glu-Leu-Ser-Leu-Glu-Leu-OH 178. H-Gly-Pro-Pro-Ile-Ser-Ile-Glu-Leu-Ser-Leu-Glu-Leu-NH2 179. H-Gly-Pro-Pro-lle-Ser-Leu-Asp-Leu-Ser-Leu-Glu-Leu-OH 1 80. H-Gly-Pro-Pro-lle-Ser-Leu-Asp-Leu-Ser-Leu-Glu-Leu-NH2 1 81. H-Gly-Pro-P ro-lIo-Ser-Phe-Asp-Leu-Ser-Leu-Glu-Leu-OH 1 82.H-Gly-Pro-Pro-lle-Ser-Phe-Asp-Leu-Ser-Leu-Glu-Leu-NH2 1 83. H-Glu-P ro-P ro-IIo-Sor-lIo-Asp-Leu-Ser-Leu-Glu-Lou-0H 1 84. H-Glu-Pro-Pro-lle-Ser-lle-Asp-Leu-Ser-Leu-Glu-Leu-NH2 185. H-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-OH 186. H-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-NH2 The following Examples illustrate the invention. The.Rf values were determined on pre-coated plates of silica gel 60 F254 (Merck) Layer thickness 0.25 mm, length 20 cm, using the following development systems: System A: n-butanol:acetic acid water (4:1:1 by volume) System B: chloroform: methanol: 32% ammonium hydroxide (65:45:20 by volume).
"Merck" is a Trade Mark.
TLC analyses were carried out at temperatures ranging from 18 to 250 C. The Rf values can therefore change by +5%.
High pressure liquid chromatography (HPLC) analysis were carried out Qn Hibar Lichrosorb R9-1 8 (5 y) columns using: Eluent A: KH2PO4 0.02 M, pH 7, 10% acetonitrile Eluent B: KH2PO4 0.02 M; pH 7, 60% acetonitrile Mobile phase: mix A and B to obtain solution containing 10% B. Increase percent of B from 10% to 60% in 25 minutes.
High voltage paper electrophoresis is carried out with a Pherograph-Original-Frankful l Type 64 apparatus on Schleicher and Schull paper No. 2317 at pH 5.8 (pyridine acetic acid water 45:5:450 by volume) 32.5 V/cm). Electrophoretic mobilities (E5.8) are given relative to glutamic acid.
Synthesis on a polymeric support can be carried out, for example, by one of the following procedures.
Procedure A. Preparation of Boc-(AA)n-(AA)n-1... (AA)1-Hydroxymethyl Polystyrene Ester Chloromethylated polystyrene resin is esterified with the first Boc-amino acid (Boc-AA1-OH) according to Gisin, Helv. Chim. Acta, 56, 1476 (1973). The polystyrene ester is treated according to schedule A for incorporation of Boc-(AA)2-OH... Boc(AA)n-OH to give the title resin.
Schedule A -1. Wash 3 times with dichloromethane.
2. Treat twice for 1 minute with trifluoroacetic acid : dichloromethane (40:60 by volume).
3. Treat for 30 minutes with trifluoroacetic acid: dichloromethane (40:60 by volume).
4. Wash 4 times with dichloroethane.
5. Treat twice for 1 minute with 10% N-methylmorpholine in dichloromethane.
6. Treat for 5 minutes with 10% N-methylmorpholine in dichloromethane.
7. Wash 8 times with dichloromethane.
8;Add or 3 equivalents of the symmetric anhydride of the corresponding amino acid derivative, prepared according to Hagenmayer and Frank, Hoppe-Seyler's Z Physiol. Chem., 353, 1973 (1972), dissolved in dichloromethane. Reaction time 0.5 to 2 hours.
9. Wash 3 times with dichloromethane.
10. Wash 3 times with isopropanol.
11. Wash 3 times with dichloromethane.
12. Test by the ninhydrin reaction according to Kaiser et al., Annal. Biochem., 34, 595 (1970). In case of incomplete reaction repeat steps 4 to 11.
Procedure B. Preparation of H-(AA),--(AA),~,... (AA)1-Hydroxyrnethyi Polystyrene Ester After introduction of the last amino acid derivative according to schedule A (procedure A), repeat steps 1 to 7 of schedule A, and wash 4 times with isopropanol.
Procedure C. Preparation of Boc-(AA)n-(AA)n-1... (AA)1-Benzhydrylamine Resin Boc-(AA)1-OH is attached to a benzhydrylamine resin via dicyclohexylcarbodiimide, as described by Pietta et al., J. Org. Chem. 39,44 (1974). Unreacted amino groups are acetylated with acetic anhyddde : pyridine : dichloromethane (2 :1:1 0 by volume). The polystyrene amide is then treated according to schedule A (procedure A) for high voltage paper electrophoresis is carried out with a Pherograph-Original-Frankfurt Type 64 apparatus on Schleicher and Schull paper No. 231 7 at pH 5.8 (pyridine:acetic acid:water 45:5:450 by volume) 32.5 V/cm. Electrophoretic mobilities (E5.6) are given relative to glutamic acid.
Synthesis on a polymeric support can be carried out, for example, by one of the following procedures.
Procedure A. Preparation of Boc-(AA)n-(AA)n-1... . . (AA)1-Hydroxymethyl Polystyrene Ester Chloromethylated polystyrene resin is esterified with the first Boc-amino acid (Boc-AA1-OH) according to Gisin, Helv. Chim. Acta, 56, 1476 (1973). The polystyrene ester is treated according to schedule A for incorporation of Boc-(AA)2-OH... Boc(AA)n-OH to give the title resin.
Schedule A 1. Wash 3 times with dichloromethane.
2. Treat twice for 1 minute with trifluoroacetic acid dichloromethane (40:60 by volume).
3. Treat for 30 minutes with trifluoroacetic acid : dichloromethane (40:60 by volume).
4. Wash 4 times with dichloroethane.
5, Treat twice for 1 minute with 10% N-methylmorpholine in dichloromethane.
6. Treat for 5 minutes with 10% N-methylmorpholine in dichloromethane.
9. Wash 8 times with dichloromethane.
8. Add 2 or 3 equivalents of the symmetric anhydride of the corresponding amino acid derivative, prepared according to Hagenmayer and Frank, Hoppe-Seyler's Z Physiol. Chem., 353, 1973 (1972), dissolved in dichloromethane. Reaction time 0.5 to 2 hours.
9. Wash 3 times with dichloromethane.
10. Wash 3 times with isopropanol.
11. Wash 3 times with dichloromethane.
12. Test by the ninhydrin reaction according to Kaiser et al., Annal. Biochem., 34,595 (1970). In case of incomplete reaction repeat steps 4 to 11.
Procedure B. Preparation of H-(AA)n-(AA)n-1.. (AA)1-Hydroxymethyl Polystyrene Ester After introduction of the last amino acid derivative according to schedule A (procedure A), repeat steps 1 to 7 of schedule A, and wash 4 times with isopropanol.
Procedure C.)reparation of Boc-(AA)n-(AA)n-1... (AA)1-Benzhydrylamine Resin Boc-(AA)1-OH is attached to a benzhydrylamine resin via dicyclohexylcarbodiimide, as described by Pietta et al., J. Org. Chem. 39,44 (1974). Unreated amino groups are acetylated with acetic anhydride;pyridine: dichloromethane (2 :1:10 by volume).The polystyrene amide is then treated according to schedule A (procedure A) for EXAMPLE 1 Pyr-Giy-Pro-Pro-lle-Ser-lle-Asp-Leu-Ser-Leu-Glu-Leu-OH Sodium Salt (1) 1 g of peptide resin deteained by procedure A with the required sequence of amino acid residues (introduced as Boc-Leu-OH, Boc-Glu(OBz1)-OH, Boc-Leu-OH, Boc-Ser(Bzl)-OH Boc-Leu-OH, Boc Asp(OBzl)-OH, Boc-lle-OH, Boc-Ser(Bzl)-OH, Boc-lle-OH, Boc-Pro-OH, Boc-Pro-OH, Boc-Gly-OH, H-Pyr OH in that ordine) was suspended for 1 hour at 0 C in 10 ml of anhydrous (distilled over 500 mg of cobalt trifluoride) hydrogen fluoride containing 1 ml of anisole. The hydrogen fluoride was removed under reduced pressure and the anisole was removed by washing with diisopropyl ether (3 x 10 ml).
The crude peptido was extracted from the resin with dimethylformamide (3 x 10 ml) and purified by gel filtration on Sephadex LH-20 using dimethylformamide as eluent, and by an exchange chromatography on DEAE-Sephadex A-25 using as eluent ammonium acetate buffer at pH 6. The product was then transformed to the sodium salt with an excess of sodium bicarbonate, desalted on Sephadex C-1 5, and lyophilized.
0.120 g of the peptide (1 ) were obtained.
RIA 0.17; RfB 0.44; E56 0.60 Clu; R, (HPLC) ca. 15' Amino acid ratio: Asp 0.99; Ser 1.94; Glu 1.96; Pro 1.96; Gly 1.01; Leu 2.96; lle 1.97.
EXAMPLE 2 Pyr-Gly-Pro-Pro-lle-Ser-lle-Asp-Leu-Ser-Leu-Glu-Leu-NH2 Sodium Salt (2) 1 g of peptide resin obtained by procedure C with the required sequence of amino acid residues (introduced as Boc-Leu-OH, Boc-Glu(OBzl)-OH, Boc-Leu-OH, Boc-Ser-(Bzl)-OH, Boc-Leu-OH, Boc Asp(OBzl)-OH, Boc-lle-OH, Boc-Ser(Bzl)-OH, Boc-lle-OH, Boc-Pro-OH, Boc-Pro-OH, Boc-Gly-OH, H-Pyr OH, in that order) was suspended for 1 hour in 10 ml of anhydrous (distilled over 500 mg of cobalt trifluoride) hydrogen fluoride containing 1 ml of anisole. The hydrogen fluoride was removed under reduced pressure and the anisole was removed by washing with diisopropyl ether (3 x 10 ml).The crude peptido was extracted from the resin with dimethylformamide (3 x 10 ml) and purified by gel filtration on Sephadex LH-20 using dimethylformamide as fluent, and by ion exchange chromatography on DEAE-Sephadex A-25 using as eluent ammonium acetate buffer at pH 6. The product was then transformed to the sodium salt with an excess of sodium bicarbonate, desalted on Sephadex G-1 5 and lyophilized.
0.130 g of the peptide (2) were obtained.
E5 , 0.30.
Amino acid ratio: Asp 0.98; Ser 1.95; Glu 1.97; Pro 1.96; Gly 1.00; Leu 2.94; lle 1.98.
EXAMPLE 3 H-Giy-Pro-Pro-lle-Ser-lle-Asp-Leu-Ser-Leu-Glu-Leu-OH Sodium Salt (1 65) 1 g of peptide resin obtained by procedure B with the required sequence of amino acid residues (introduced as Boc-Leu-OH, Boc-Glu(OBzl)-OH, Boc-Leu-OH, Boc-Ser-(Bzl)-OH, Boc-Leu-OH, Boc Asp(OBzl)-OH, Boc-lle-OH, Boc-Ser(Bzl)-OH, Boc-lle-OH, Boc-Pro-OH, Boc-Pro-OH, Boc-Gly-OH in that order) was suspended for 1 hour at OOC in 10 ml of anhydrous (distilled over 500 mg of cobalt trifluoride) hydrogen fluoride containing 1 ynl of anisole. The hydrogen fluoride was removed under reduced pressure and:the anisole was removed by washing with diisopropyl ether (3 x 10 ml).The curde peptide was extracted from the resin with dimethylformamide (3 x 10 -mI) and purified by gel filtration on Sephadex LH-20 using dimethylformamide as eluent and by ion exchange chromatograph on CH-Sephadex C-25 using as eluent ammonium acetate buffer at pH 4. The product was then transformed to the sodium salt with an excess of sodium bicarbonate, desalted on Sephadex Ct-1 5 and lyophilized.
0.110 g of the peptide (16.5) were obtained.
RfA 0.16, RfB 0.44, E58 0;34; Rt (HP'LC) Ca. 17' Amino acid ratio: Asp 0.98; Ser 1.97; Glu 1.00; Pro 1.99; Gly 0.99 Leu 2.97; lle 1.98.
EXAMPLE 4 H-Gly-Pro-Pro-lle-Ser-lle-Asp-Leu-Sers Leu-Glu-Leu-NH2 Sodium Salt (166) 1 g of peptide resin obtained by procedure D with the required sequence of amino acid residues (introduced as Boc-Leu-OH, Boc-Glu (OBzl)-OH, Boc-Leu-OH, Boc-Ser(Bzl)-OH, Boc-Leu-OH, Boc Asp(OBzl)-OH, Boc-lle-OH, Boc-Ser(Bzl)-OH, Boc-lle-OH, Boc-Pro-OH, Boc-Pro-OH, Boc-Gly-OH in that order) was suspended for 1 hour at OOC in 10 ml of anhydrous (distilled over 500 mg of cobalt trifluoride) hydrogen fluoride containing 1 ml of anisole. The hydrogen fluoride wes removed under reduced pressure and the anisole was removed by washing with diisopropyl ether.The crude peptido was extracted from the resin with dimethylformamide (3 x 10 ml) and purified by gel filtration on Sephadex LH-20 using dimethylformamide as eluent and by ion exchange chromatography on CH Sephadex C-25 using as eluent ammonium acetate buffer at pH 4. The product was then transformed to the sodium salt with an excess of sodium bicarbonate, desalted on Sephadex G-1 5 and lyophilized.
0.110 g of the peptide (1 66) were obtained.
ES.e." 0 Amino acid ratio: Asp 0.98; Ser 1.97; Glu 0.99; Pro 1.97; Gly 1.00; Leu 2.96; lle 1.96.
EXAMPLE 5 Preparation of H-Gln-Glu-Pro-Pro-lle-Ser-Leu-Asp-Leu-Thr-Phe-OH Sodium Salt (89) 1 g of peptide-resin obtained by procedure B with the required sequence of amino acid residues (Boc-Phe-OH, Boc-Thr(Bzl)-OH, Boc-Leu-OH, Boc-Asp (OBzl)-OH, Boc-Leu-OH, Boc-Ser(Bzl)-OH, Boc lle-OH, Boc-Pro-OH, Boc-Pro-OH, Boc-Glu(OBzl)-OH, Boc-Gln-OH, in that order) was suspended for 1 hr at OOC in 1.0 ml of anhydrous (distilled over 500 mg of cobalt trifluoride) hydrogen fluoride containing 1 ml of anisole. The hydrogen fluoride was removed under reduced pressure and the anisole was removed by washing with diisopropyl ether (3 x 10 ml).The crude peptide was extracted from the resin with dimethylformamide (3 x 10 ml) and purified by gel filtration on Sephadex LH-20 using dimethylformamide as eluent and by ion exchange chromatography on CH-Sephadex C-25 using as eluent ammonium acetate at pH 4. The product was then converted to the sodium salt with an excess of sodium bicarbonate, desalted on Sephadex G-1 5 and lyophilized.
0.130 g of the peptide (89) were obtained.
RfA 0.22, Rfs 0.45, E58 0.75 Rt (HPLC) Ca. 12' Amino acid ratio: Asp 0.99; Thr 0.88; Ser 0.96, Glu 1.98, Pro 1.98, Leu 2.00; Ile 0.98; Phe 0.99 In similar way the following compounds were prepared: VI) Pyr-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser-Leu-Glu-OH Sodium Salt (45) E5,8 0.63 Amino acid ratio: Asp 0.98; Ser 1.96; Glu 1.98; Pro 1.99; Gly 1.00; Leu 1.97; lle 1.98.
VII) Pyr-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser-Leu-Glu-NH2 Sodium Salt (46) E5.8 0.30.
Amino acid ratio: Asp 0.99; Ser 1.95; Glu 1.97; Pro 1.98; Gly 1.00; Leu 1.98; lle 1.97.
VIII) Pyr-Gly-P ro-Pro-ile-Ser-lle-Asp-Leu-Ser-Leu-OH Sodium Salt (69) E5.8 0.32 Amino acid ratio: Asp 0.98; Ser 1.97; Glu 0.98; Pro 1.98, Gly 1.00, Leu 1.99.
IX) Pyr-Gly-Pro-Pro-lle-Ser-lle-Asp-Leu-Ser-Leu-NH2 Sodium Salt (70) E5.8 0.15.
Amino acid ratio: Asp 0.99, Ser 1.98; Glu 0.99; Pro 1.97; Gly 1.00; Leu 1.99; lle 1.99; X) Pyr-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser-OH Sodium Salt (91) E5.ss 0.35.
Amino acid ratio: Asp: 0.97; Ser 1.96; Glu 0.97; Pro 1.96; Gly 0.99; Leu 1.00; lie 1.98.
XI) Pyr-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser-NH2 Sodium Salt (92) E5.8 0.16.
Amino acid ratio: Asp 0.98, Ser 1.97; Glu 1.01; Pro 1.97; Gly 1.00; Leu 1.00; lle 1.97.
XII) H-Gly-Pro-Pro-lle-Ser-lle-Asp-Leu-Ser-OH Sodium Salt (111) E5,8 0.18.
Amino acid ratio: Asp 0.99; Ser 1.98; Pro 1.99; Gly 1.01; Leu 1.00; Ile 1.99.
XIII) H-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser-NH2 Sodium Salt (112) E5.8 ca. O.
Amino acid ratio: Asp 0.98; Ser 1.98; Pro 1.98; Gly 0.99; Leu 1.00; lie 1.98.
XIV) H-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser-Leu-OH Sodium Salt(127) E580.15 Amino acid ratio: Asp 0.99; Ser 1.97; Pro 1.99; Gly 1.00; Leu 1.99; Ile 1.98.
XV) H-Gly-Pro-Pro-lle-Ser-ile-Asp-Leu-Ser-Leu-NH2 Sodium Salt (128) E5.8 ca. O.
Amino acid ratio: Asp 0.98; Ser 1.97; Pro 1.97; Gly 1.00; Leu 2.00; Ile 1.99.
XVI) H-Gly-Pro-Pro-lle-Ser-lle-Asp-Leu-Ser-Leu-Glu-OH Sodium Salt (145) E5.8 0.32.
Amino acid ratio: Asp 0.99; Ser 1.98; Glu 0.97; Pro 1.98; Gly 1.00; Leu 2.01; lIe 1.99.
XVII) H-Gly-Pro-Pro-lle-Ser-lle-Asp-Leu-Ser-Leu-Glu-NH2 Sodium Salt (146) E5.B 0.16.
Amino acid ratio: Asp 0.98; Ser 1.98; Glu 0.98; Pro 1.99; Gly 1.00; Leu 1.98; lle 1.98.
XVIII) Pyr-Gly-Pro-Pro-lle-Ser-lle-Asn-Leu-Ser-Leu-Glu-OH Sodium Salt (13) E5 8 0.28.
Amino acid ratio: Asp 0.99; Ser 1.99; Glu 1.99; Pro 1.98; Gly 1.00; Leu 2.97; lle 1.97.
XIX) Pyr-Gly-Pro-Pro-lle-Ser-lle-Asn-Leu-Ser-Leu-Glu-Leu-NH2 Sodium Salt (11) E5.8 0.14.
Amino acid ratio: Asp 0.99; Ser 1.97; Glu 1.97; Pro 1.99; Gly 1.00; Leu 2.98; lle 1.99.
XX) Pyr-Gly-Pro-Pro-lle-Ser-ile-Asp-Leu-Ser-Leu-Gln-Leu-OH Sodium Salt (5) E5 8 0.28.
Amino acid ratio: Asp 0.97; Ser 1.98; Glu 1.96; Pro 1.97; Gly 1.00; Leu 2.97; lle 1.97.
XXI) Pyr-Gly-Pro-Pro-lle-Ser-lle-Asp-Leu-Ser-Leu-Gln-Leu-NH2 Sodium Salt (6) E58 0.14.
Amino acid ratio: Asp 0.98; Ser 1.97; Glu 1.97; Pro 1.99; Gly 1.00; Leu 3.01; lle 1.99.
XXII) Pyr-Gly-Pro-Pro-lle-Ser-lle-Asp-Leu-Ser-Leu-Giu-Phe-OH Sodium Salt (3) RfA 0.16; Rf5 0.42; E58 0.60.
Amino acid ratio: Asp 0.99; Ser 1.95; Glu 1.99; Pro 1.98; Gly 1.00; Leu 1.97; lle 1.98; Phe 1.00.
XXIII) Pyr-Giy-Pro-Pro-lle-Ser-lle-Asp-Leu-Ser-Leu-Glu-Phe-NH2 Sodium Salt (4) E5.a 0.30.
Amino acid ratio: Asp 0.98; Ser 1.98; Glu 1.96; Pro 1.98; Gly 1.01; Lou 1.99; lle 1.98; Phe 1.00.
EXAMPLES 24 TO 186 Using corresponding starting materials and by way of corresponding intermediates, the peptides identified above by the numbers 7 to 12, 15to44,47 to 68,71 to 90, 93 to 110, 1 13 to 126,129 to - 144. 147 to 1 86 are obtained analogously to the aforegoing Examples.

Claims (4)

1. A peptide of the general formula: X-A-Pro-Pro-lle-Ser-B-C-Leu-D-E-F-G-W wherein: X represents a hydrogen atom, a terminal nitrogen protecting group of acyl, aromatic urethane, alkyl, aralkyl or aliphatic urethane type or a residue of a neutral L-amino acid, the free amino group of the residue optionally being protected by a terminal nitrogen atom protecting group of the type cited above, A represents an L-amino acid residue or a glycine residue; B represents a neutral L-amino acid residue; C represents a neutral L-amino acid residue or an acidic L-amino acid residue; D represents an L-amino acid residue which contains in the side chain an alcoholic hydroxy group, which may be free or protected by a conventional hydroxy protecting group; E represents a valence bond or a neutral L-amino acid residue;; F represents a valence bond or an L-amino acid residue; G represents a valence bond or a neutral L-amino acid residue; W represents a hydroxy group, an amino group or a group of the formula OR, NHR, NR2 or NH-NH R' wherein R represents a straight chain, branched chain or cyclic (including fused or bridged ring) alkyl group having up to 1 1 carbon atoms, and being substituted or unsubstituted, a phenyl group or an aralkyl group having from 6 to 8 carbon atoms; and R' represents a hydrogen atom, any of the groups which R may represent, an alkenyl group having from 3 to 8 carbon atoms; a straight chain, branched chain or cyclic aliphatic acyl group having from 1 to 11 carbon atoms, unsubstituted or substituted by hydroxy or an amino group or a halogen atom, an aromatic acyl group, unsubstituted or substituted by a hydroxy or amino group or a halogen atom, a straight chain, branched chain or cyclic aliphatic urethane type group having from 3 to 11 carbon atoms, or an aromatic urethane type group; or a pharmaceutically acceptable salt thereof.
2. A peptide according to claim 1, wherein, X represents hydrogen atom, a formyl, acetyl, trifluoroacetyl, propionyl, bonzoyl, benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4- methoxybenzyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl, 2-bromobenzyloxycarbonyl, 9- fluorenylmethoxycarbonyl, methoxy-cr,a'-dimethylbenzyloxywarbonyl, t-butoxycarbonyl, 1 - methyl-cyclobutoxyca rbonyl, adamantyloxycarbonyl, isobornyloxycarbonyl, trityl, benzyl, methyl or isopropyl group or a Pyr, Gln, Pro or 2-oxo-L-pipecolic acid residue, A represents a Glu, Gln or Gly residue, B represents an lle, Leu, Nle, Val or Phe residue, C represents an Asn, Gln, Asp or Glu residue, D represents a Ser, Hse and Tlr residue and if the hydroxy group of the residue represented by D is protected, it is protected by a t-butyl, trityl, benzyl, 2,4-dichlorobenzyl, benzyloxycarbonyl, 2bromobenzyloxycarbonyl, tetrahydropyranyl, t-butoxyca rbonyl, formyl, acetyl, trifluoroacetyl, propionyl or benzoyl group, E represents a Phe, Leu or Nle residue or a valence bond, F represents a Glu, Gln or His residue or a valence bond, G represents a Leu or Phe residue or a valence bond, and W represents a hydroxy group, an amino group or a group of the formula OR, NHR, NR2 or NH-NH R' wherein R represents a methyl, ethyl, n-propyl, isopropyl, n-butyl, s-butyl, isobutyl, t-butyl, 2,2,2- trifluoroethyl, cyclohexyl, adamantyl, phenyl, bonzyl or phenethyl group and R' represents a hydrogen atom or an allyl, pentenyl, formyl, acetyl, trifluoroacetyl, propionyl, butyryl, adamantylcarbonyl, benzoyl, phenylacetyl, cinnamyl, benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl, 2-bromobenzyloxycarbonyl, 9-fluorenylmethoxycarbonyl, 3,5 dimethoxy-ct,a'-dimethyibenzyloxywarbonyl, t-butoxycarbonyl group, or any of the groups specifically named in this claim as values for R; or a pharmaceutically acceptable salt thereof.
3. Any of the peptidos numbered herein from 1 to 186, or a pharmaceutically acceptable salt thereof.
4. A pharmaceutical composition comprising a peptido according to any preceding claim or a pharmaceutically acceptable salt thereof in admixture with a pharmaceutically acceptable diluent or carrier.
GB08309547A 1982-04-13 1983-04-08 Peptides with sauvagine-like activity Expired GB2118190B (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1986001209A3 (en) * 1984-08-02 1986-03-27 Boehringer Mannheim Gmbh New p-phenylenediamine peptides and reactance containing them for the determination of proteases of the blood coagulation system
EP0122798A3 (en) * 1983-04-14 1986-09-03 The Salk Institute For Biological Studies Rcrf and analogs

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0122798A3 (en) * 1983-04-14 1986-09-03 The Salk Institute For Biological Studies Rcrf and analogs
WO1986001209A3 (en) * 1984-08-02 1986-03-27 Boehringer Mannheim Gmbh New p-phenylenediamine peptides and reactance containing them for the determination of proteases of the blood coagulation system

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