DE19732944A1 - Derivatives of calcitonin-gene-related peptide fragment - Google Patents

Abstract

New peptides derived from calcitonin-gene-related peptide (rCGRP- alpha ) (positions 27-37) sequence H-FVPTNVGSEAF-NH2 where: (i) 1-3 of the amino acids in the sequence FVPTNVGSE are replaced with K, Y, A, F, P, Hyp, S, L, Q, D, H, Acp, Pac, Tic, Hop, Aib, I or G; or (ii) the sequence FVPTNVGS is replaced with NFVPRSKISP, NVAPRSKISP, NVAPTNVGS or ILSSTNVGS; and/or (iii) 1-8 of the amino acids in the sequence FVPTNVGSE are deleted; and (iv) one amino acid in the sequence FVPTNVGSEAF can be a D-amino acid; and (v) the N-terminal amino group can be substituted by a protecting group R selected from 3,3-diphenylpropionyl, 2-propylpentanoyl, 3,5-dichlorophenoxyacetyl, 1-adamantylacetyl, 3,5-dibromo-4-hydroxyphenylalanyl, 4-(4-benzhydryl-1-piperazinyl)-1,5-dioxo-3,3-(tetramethylene)-pentyl or 5,11-dihydro-6-oxo-6H-dibenzb,eazepin-11-carbonyl. The peptide H-YVPTNVGSEAF-NH2 is excluded.

Description

The present invention relates to new partial sequences of the r-CGRP-alpha-27-37

HF ²⁷ -V ²⁸ -P ²⁹ -T ³⁰ -N ³¹ -V ³² -G ³³ -S ³⁴ -E ³⁵ -A ³⁶ -E ³⁷ -NH ₂ (I)

derived peptides, characterized in that in formula I

• (i) one, two or three of the amino acids of positions 27 to 35 independently of one another by amino acids from the group
  K, Y, A, F, P, Hyp, S, L, Q, D, H, Acp, Pac, Tic, Hop, Aib, I and G
  are replaced or
• (ii) the amino acids of positions 27 to 34 by the sequences
  NFVPRSKISP ³⁴ , NVAPRSKISP ³⁴ , NVAPTNVGS ³⁴ or ILSSTNVGS ³⁴
  are replaced or / and
• (iii) one to eight of the amino acids of positions 27 to 35 are replaced by single bonds and
• (iv) additionally an amino acid from positions 27 to 37 Can be configured.

where the N-terminal NH ₂ groups contained in the compounds may be substituted by the radical R and R represents a protective radical or the 3,3-diphenylpropionyl, 2-propylpentanoyl, 3,5-dichlorophenoxyacetyl, 1-adamane tylacetyl -, 3,5-dibromo-4-hydroxyphenylalanyl-, 5- (4-benzhydrylpiperazin-1-yl) -1,5-dioxo-3,3-tetramethylene-pentyl- or 5,11-dihydro-6 (6H ) -oxodibenz [b, e] azepin-11-ylcarbonyl group,
with the exception of the compound H-YVPTNVGSEAF-NH _2,
and their salts, in particular for pharmaceutical use, their physiologically tolerable salts with inorganic or organic acids or bases, medicaments containing these compounds, their use and process for their preparation.

The compounds according to the invention have valuable pharmacolo properties based on their selective CGRP-antago characteristics decline.

The protective residues mentioned in the definitions above and below are to be understood as the protective groups familiar from peptide chemistry, in particular
a phenylalkoxycarbonyl group with 1 to 3 carbon atoms in the alkoxy part, optionally substituted by a halogen atom, by a nitro or phenyl group, by one or two methoxy groups,
for example the benzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-meth oxybenzyloxycarbonyl, 2-chloro-benzyloxycarbonyl, 3-chloro-benzyloxycarbonyl, 4-chloro-benzyloxycarbonyl, 4 Biphenylyl-α, α-dimethyl-benzyloxycarbonyl or 3,5-dimethoxy-α, α-dimethyl-benzyloxycarbonyl group,
an alkoxycarbonyl group with a total of 1 to 5 carbon atoms in the alkyl part,
for example the methoxycarbonyl, ethoxycarbonyl, n-propoxycarbonyl, isopropoxycarbonyl, n-butoxy carbonyl-1-methylpropoxycarbonyl, 2-methylprop oxycarbonyl or tert.butyloxycarbonyl group,
the allyloxycarbonyl, 2,2,2-trichloro (1,1-dimethylethoxy) car bonyl or 9-fluorenylmethoxycarbonyl group or
the formyl, acetyl or trifluoroacetyl group.

Unless otherwise stated, the divalent amino acid residues specified in the definitions above and below are L-configured and have the customary one- or three-letter code, as described, for example, in US Pat. B. in Eur. J. Biochem. 138, 9-37 (1984) has the following meanings:

Preferred peptides derived from the r-CGRP-alpha-27-37 partial sequence according to the present invention are characterized in that in formula (I)

• (i) an amino acid of positions 27 to 35 by one of the Amino acids K, Y, A, P or D is replaced or
• (ii) F ^{27 is} replaced by Y and in addition one of the amino acids from positions 28 to 35 is replaced by A, F, Hyp, S, L, Q, D, K or H or
• (iii) F ^{27 is} replaced by Y and in addition one of the amino acids of positions 27 to 37 is D-configured or
• (iv) an amino acid from positions 27 to 35 by D er is set and in addition another amino acid Positions 27 to 35 are replaced by Q or P or  
• (v) an amino acid from positions 27 to 35 by D er is set and two additional neighboring ones Amino acids of positions 27 to 35 by A-H, A-F, P-F, Acp-F, Pac-F, Tic-F, Hyp-F, Hop-F or Aib-F are replaced or
• (vi) one or both amino acids of positions 27 and 28 are replaced by a single bond, an additional one Amino acid of positions 29 to 35 is replaced by D. and two other adjacent amino acids of the positions 29 to 35 are replaced by A-F or
• (vii) the two amino acids of positions 27 and 28 a single bond are replaced and an additional one Amino acid of positions 29 to 35 by A or two adjacent amino acids from positions 29 to 35 A-F are replaced or
• (viii) two adjacent amino acids from positions 29 to 35 are replaced by A-F or P-F or
• (ix) F ^{27 is} replaced by a single bond, it being possible for an amino acid from positions 30 to 35 to be replaced by P, I, L, G, Aib or D and, independently thereof, two further adjacent amino acids from positions 30 to 35 to be replaced by AF or
• (x) the amino acids of positions 27 and 28 or 27 to 29 are replaced by a single bond, with additional Lich an amino acid from positions 31 to 35 by Aib or A or two adjacent amino acids of the positions 31 to 35 can be replaced by A-F or
• (xi) the amino acids of positions 27 and 28 by a Single bond are replaced, an additional amino acid of positions 31 to 35 by D and two more be  neighboring amino acids from positions 31 to 35 A-F are replaced or
• (xii) F ^{27 is} replaced by Y and one to eight of the amino acids in positions 28 to 35 are omitted.

where the N-terminal NH ₂ contained in the compounds

Groups can be substituted by the radical R and R represents a protective radical or the 3,3-diphenylpropionyl, 2-propylpentanoyl, 3,5-dichlorophenoxyacetyl, 1-adamane tylacetyl, 3,5-dibromo-4-hydroxyphenylalanyl -, 5- (4-Benzhydrylbiperazin-1-yl) -1,5-dioxo-3,3-tetramethylene-pentyl- or 5,11-dihydro-6 (6H) -oxodibenz [b, e] azepin-11 -ylcarbonyl group means
with the exception of the compound H-YVPTNVGSEAF-NH ₂

,
and their salts.

Particularly preferred peptides according to the present invention derived from the r-CGRP-alpha-27-37 partial sequence are characterized in that in formula (I)

• (i) an amino acid of positions 34 or 35 by K is replaced or
• (ii) F ^{27 is} replaced by Y or
• (iii) an amino acid of positions 28, 31 or 34 by P is replaced or
• (iv) an amino acid of positions 30 or 31 by D is replaced or
• (v) F ^{27 is} replaced by Y and additionally either one of the amino acids of positions 28 or 34 is replaced by A or
  the amino acid of position 35 by F or
  the amino acid of position 29 by Hyp or
  one of the amino acids of positions 29 to 31 by L or D or
  one of the amino acids of positions 34 or 35 is replaced by K, H, Q or L.

or

• (vi) the amino acid of position 31 is replaced by D and an additional amino acid from positions 34 or 35 by Q or P or the amino acids of the Positions 34 and 35 by A-H, A-F, P-F, Acp-F, Pac-F, Tic-F, Hyp-F, Hop-F or Aib-F are replaced or
• (vii) one or both amino acids of positions 27 and 28 are replaced by a single bond, additionally the Amino acid of position 31 by D and the amino acids of items 34 and 35 are replaced by A-F or
• (viii) the two amino acids of positions 27 and 28 a single bond are replaced and an additional one Amino acid of positions 34 or 35 by A or both Amino acids of positions 34 and 35 replaced by A-F are or
• (ix) the amino acids of positions 34 and 35 by A-F or P-F are replaced or  
• (x) F ^{27 is} replaced by a single bond and additionally an amino acid of positions 31 or 34 by P or Aib or
• (xi) F ^{27 is} replaced by a single bond and V ²⁸ on the N-terminal amino group by a 3,3-diphenylpropionyl, 2-propylpentanoyl, 3,5-dichlorophenoxyacetyl, 1-adamantylacetyl, 3.5 -Dibromo- 4-hydroxyphenylalanyl- or 5- (4-benzhydryl-piperazin-1-yl) -1,5-dioxo-3,3-tetramethlen-pentyl group is substituted, the amino acid of position 31 being substituted by D and the amino acids of positions 34 and 35 can be replaced by AF or
• (xii) the amino acids of positions 27 and 28 are replaced by a single bond and P ²⁹ on the amino group by a 3,3-diphenylpropionyl, 3,5-dichlorophen oxyacetyl, 1-adamantylacetyl or 5- (4-benzhydryl piperazine -1-yl) -1,5-dioxo-3,3-tetramethylene-pentyl group is substituted, wherein in addition the amino acid of position 34 can be replaced by Aib or
• (xiii) the amino acids of positions 27 and 28 are replaced by a single bond, P ²⁹ on the amino group is substituted by a 3,5-dichlorophenoxyacetyl group, the amino acids of positions 34 and 35 are replaced by AF and additionally the amino acid of the position 31 can be replaced by D or
• (xiv) the amino acids of positions 27 to 29 are replaced by a single bond and T ³⁰ on the amino group is substituted by a 3,5-dichlorophenoxyacetyl or 1-adamane tylacetyl group.

where the N-terminal NH ₂ contained in the compounds

Groups may be substituted by the radical R and R represents a protective radical, for example the acetyl radical, with the exception of the compound H-YVPTNVGSEAF-NH ₂

,
and their salts.

The following may be mentioned as particularly preferred compounds:

and their salts.

The compounds of the invention can be by generally known methods of peptide chemistry, such as. B. in Houben-Weyl, Methods of Organic Chemistry, Vol. 15/2, or preferably after solid-phase peptide synthesis (e.g.: B. Barany, RB Merrifield in The Peptides, Analysis, Synthesis, Biology, Vol. 2 , 2-284 [1980], Academic Press, New York, or RC Sheppard, Int. J. Pept. Prot. Res. 21, 118 [1983]) or equivalent known methods. As amino protecting groups, those described in Houben-Weyl, Methods of Organic Chemistry, Vol. 15/1, are used before urethane protecting groups such as, for. B. the fluorenyl methoxycarbonyl or the tert-butyloxycarbonyl protective group applied. To prevent side reactions, any functional groups present in the side chains of the amino acids are protected by suitable protective groups (see, for example: GB Fields et al., Int. J. Peptide Protein Res 35, 161 (1990); TW Greene , Protective Groups in Organic Synthesis) are additionally protected. Arg (NO ₂ ), Arg (Mtr), Arg (di-Z), Arg (Pmc), Tyr (t-Bu), Tyr (Bzl), Tyr (Br-Z), Ser (t- Bu), Ser (Bzl), Glu (t-Bu), Glu (Bzl), Lys (Boc) Lys (Z), His (Trt), His (Bum), His (Dnp), His (Z), Thr (t-Bu), Thr (Bzl), Cys (Acm), Cys (4-Me-Bzl), Cys (t-Bu), Asp (t-Bu) Asp (Bzl), Orn (Boc), Orn ( Z), Cys (Trt), Asn (Trt), Asn (Tmob), Asn (Mbh), Gln (Trt), Gln (Tmob), Gln (Mbh) and Lys (Cl-Z).

For the synthesis of the compounds of the invention after the festival phase synthesis are suitable as anchor groups all those  that fully when the peptide is split off from the polymeric carrier provide protected peptide amides. After splitting off the amino protecting group of the amino acid coupled to the resin with a suitable reagent, e.g. B. with trifluoroacetic acid in dichloro methane in the case of the Boc group or a 20-50% solution of Piperidine in DMF in the case of the Fmoc group, will be suitable protected amino acids coupled. This cycle will follow run through each other until the desired resin-bound Peptide is formed.

The methods known in peptide chemistry can be used for coupling (see Houben-Weyl, Methods of Organic Chemistry, Vol. 15/2) be applied. Carbodiimides, such as e.g. B. dicyclohexylcarbodiimide, diisopropylcarbodiimide or Ethyl (3-dimethylaminopropyl) carbodiimide or O- (1H-benzo triazol-1-yl) -N, N, N ', N'-tetramethyluronium hexafluorophosphate (HBTU) or tetrafluoroborate (TBTU) or 1H-benzotriazol-1-yl-oxy-tris- (di methylamino) phosphonium hexafluorophosphate (BOP). By adding 1-hydroxybenzotriazole (HOBt) or 3-hydroxy-4-oxo-3,4-di hydro-1,2,3-benzotriazine (HOObt) can be used if necessary, suppresses the racemization or the reaction gene speed can be increased. The couplings become normal usually with a 4- to 10-fold excess of N-protected Amino acid and coupling reagent in solvents such as dichlor methane, dimethylformamide (DMF), N-methylpyrrolidone (NMP), Dimethylacetamide (DMA) or mixtures of these are carried out. The course of the coupling reaction is determined by the Kaiser test (E. Kaiser et al., Anal. Biochem. 34, 595 [1970]) or by the TNBS test tracked. If incomplete acylation is determined, the clutch is complete repeated. The solid phase synthesis can be done manually as well done automatically with the help of a peptide synthesizer.

Solid phase syntheses are preferred using the 4- (2 ', 4'-dimethoxyphenyl (Fmoc-aminomethyl) phenoxy linker, Fmoc amino protecting groups and the automatic synthesizer from ABI (430A) or the automatic multiple synthesizer SYRO  (Bochum). There are simple clutches when activated with HOBt / TBTU / DIPEA (1: 1: 1.5) and 10-fold excess of the N-ge protect amino acid or double couplings with DIC / HOBt-Akti vation and 5- to 10-fold excess of N-protected amino acid performed. The resin is applied after each coupling step washed with DMF and isopropanol.

After the peptide has been built up, the polymer carrier also contains the prin zip split off known reagents, namely in In the case of peptides constructed according to the Boc strategy for example with liquid hydrogen fluoride, in the case of after the Fmoc strategy peptides obtained with Tri fluoroacetic acid. The side chain protection groups split off.

Usually when splitting off the side chain Protecting groups as cation scavengers substances such as phenol, cresol, Anisole, thiocresol, thioanisole, indole, dimethyl sulfide or Ethyl methyl sulfide or a mixture of these substances Hydrogen fluoride or trifluoroacetic acid added.

The raw products obtained are cleaned by means of gel chromatography, e.g. B. on Sephadex G25 with 1% or 5% Acetic acid and / or by means of preparative RP-HPLC with methanol or Acetonitrile-water gradients with addition of 0.05 to 2% Trifluoroacetic acid. Cations can also be used for cleaning exchangers based on Sephadex or polystyrene can be used.

The new peptides obtained in this way can, if desired then in their addition salts with inorganic or organic acids or bases, especially for pharma Zeutische application in their physiologically acceptable salts, convict. For example, sodium salts, potassium salts or salts with cyclohexylamine, ethanolamine, diethanolamine, Triethanolamine, N-methylglucosamine, arginine or lysine in Consideration. Examples of acids are hydrochloric acid, bromine hydrochloric acid, phosphoric acid, nitric acid, sulfuric acid,  Methanesulfonic acid, fumaric acid, succinic acid, lactic acid, Citric acid, tartaric acid, maleic acid, p-toluenesulfonic acid, Called acetic acid, mandelic acid and malic acid.

The compounds of the invention and their physiologically ver inert salts have CGRP-antagonistic properties and show good affinities in CGRP receptor binding studies. The Compounds point in the phar described below macological test systems CGRP-antagonistic properties on.

To demonstrate the affinity of the compounds according to the invention to human CGRP receptors and their antagonistic egg The following experiments were carried out:  

Binding studies with the human CGRP receptor expressing SK-N-MC cells

SK-N-MC cells advertise cultivated in "Dulbecco's modified Eagle Medium". The medium of confluent cultures is removed. The cells are washed twice with PBS buffer (Gibco 041-04190 M), detached by adding PBS buffer, mixed with 0.02% EDTA, and isolated by centrifugation. After re-suspension in 20 ml "Balanced Salts Solution" [BSS (in mM): NaCl 120, KCl 5.4, NaHCO ₃ 16.2, MgSO ₄ 0.8, NaH ₂ PO ₄ 1.0, CaCl ₂ 1.8, D-Glucose 5.5, HEPES 30, pH7.40], the cells are centrifuged twice at 100 × g and resuspended in BSS. After the cell number has been determined, the cells are homogenized using an Ultra-Turrax and centrifuged for 10 minutes at 3000 × g (4 ° C.). The supernatant is discarded and the pellet in Tris buffer (10 mM Tris, 50 mM NaCl, 5 mM MgCl ₂ , 1 mM EDTA, pH 7.40), enriched with 1% bovine serum albumin and 0.1% bacitracin, recentrifuged and resuspended (1 ml / 1,000,000 cells). The homogenate is frozen at -80 ° C. Under these conditions, the membrane preparations are stable for more than 6 weeks.

After thawing, the homogenate is diluted 1:10 with assay buffer (50 mM Tris, 150 mM NaCl, 5 mM MgCl ₂ , 1 mM EDTA, pH 7.40) and homogenized for 30 seconds with an Ultra-Turrax. 230 μl of the homogenate are incubated for 180 minutes at room temperature with 50 pM ¹²⁵ I-iodotyrosyl-calcitonin gene-related peptides (Amersham) and increasing concentrations of the test substances in a total volume of 250 μl. The incubation is ended either by centrifugation at 2900 × g for ten minutes or by rapid filtration through GF / B glass fiber filters treated with polyethyleneimine (0.1%) using a cell harvester. The radioactivity bound to protein is determined with the aid of a gamma counter. The bound radioactivity after the presence of 1 μM human CGRP-1 during the incubation is defined as non-specific binding.

The concentration-binding curves are analyzed with the help a computer-aided non-linear curve fitting.

In the test described, the compounds according to the invention show good IC ₅₀ values, mostly ≦ 10,000 nM.

Because of their pharmacological properties are suitable the compounds of the invention and their salts with phy Siologically compatible acids or bases for acute and prophylactic treatment of headaches, esp special migraine or cluster headache. Continue to be the compounds according to the invention also influence the fol diseases positive: non-insulin-dependent diabetes mellitus ("NIDDM"), cardiovascular diseases, diseases the skin, especially thermal and radiation-related skin damage including sunburn, inflammatory diseases, e.g. B. inflammatory joint diseases (arthritis), inflammatory lun genetic diseases, allergic rhinitis, asthma, diseases, those with excessive vasodilation and thereby associated with reduced tissue blood flow, e.g. B. Shock and sepsis, as well as morphine tolerance. In addition zei the compounds according to the invention have a soothing effect to pain in general.

The necessary to achieve a corresponding effect Dosage is expedient for intravenous or sub cutan administration 0.001 to 30 mg / kg body weight, preferably 0.1 to 10 mg / kg body weight, and for oral, nasal or inhalation 0.1 to 100 mg / kg body weight, preferably 1 to 100 mg / kg body weight, 1 to 3 times a day.

For this purpose, the compounds produced according to the invention, if appropriate in combination with other active substances, such as. B. antiemetics, prokinetics, neuroleptics, antidepressants, neurokinin antagonists, anticonvulsants, histamine H1 receptor antagonists, antimuscarinics, β-blockers, α-agonists and α-antagonists, ergot alkaloids, weak analgesics, non-steroidal anti-inflammatory drugs, calcium corticosteroids , 5-HT _1D agonists or other anti-migraine agents, together with one or more inert customary carriers and / or diluents, e.g. B. with corn starch, milk sugar, cane sugar, microcrystalline cellulose, magnesium stearate, polyvinylpyrrolidone, citric acid, tartaric acid, water, water / ethanol, water / glycerol, water / sorbitol, water / polyethylene glycol, propylene glycol, cetyl stearyl alcohol, carboxymethyl cellulose or hard fat content or their suitable mixtures, incorporated into conventional pharmaceutical preparations such as tablets, dragées, capsules, powders, suspensions, solutions, metered dose aerosols or suppositories.

For the above-mentioned combinations, there are therefore further active substances, for example meloxicam, ergotamine, dihydroergotamine, metoclopramide, domperidone, diphenhydramine, cyclizine, promethazine, chlorpromazine, dexamethasone, flunarizine, dextropropoxyphene, meperidine, propranolol, nadolol, dinophenolamine, clenololamine, clenololamine, clonolamine Carbamazepine, phenytoin, valproate, amitrypiline, lidocaine, diltiazem or sumatriptan and other 5-HT _1D agonists such as B. Naratriptan, Zolmitriptan, Avitriptan, Rizatriptan and Eletriptan. The dose for these active substances is expediently 1/5 of the usually recommended lowest dose up to 1/1 of the normally recommended dose, for example 20 to 100 mg sumatriptan.

Another object of the invention is the use of the compounds according to the invention as valuable tools for the production and purification (affinity chromatography) of antibodies and, after suitable radioactive labeling, for example by direct labeling with 125 _I or 131 _I or by tritizing suitable precursors, for example by Replacement of halogen atoms with tritium, in RIA and ELISA as says and as diagnostic and analytical tools in neurotransmitter research.

The following examples are intended to illustrate the invention purify:

Preliminary remarks Analytics

The compounds were determined by RP-HPLC and / or capillary zone electrophoresis checked for purity. There was one Amino acid analysis using HPLC after gas phase hydrolysis and PTH derivatization and / or on a gas chromatograph on chiral Additional racemization control column performed. Electrospray mass spectra (API III, Sciex) added for characterization.

The sequence was determined using gas phase sequencing.

The abbreviations used for the amino acids correspond to the usual one or Three-letter code as it is e.g. B. in Eur. J. Biochem. 138, 9-37 (1984). The amino acid residues written with capital letters are L-configured, the amino acid residues marked with lower case letters are D-configured. The other abbreviations are explained below.
Acm = acetamidomethyl
Boc = tert-butyloxycarbonyl
t-Bu = tert-butyl
Bum = tert-butyloxymethyl
Bzl = benzyl
Cl-Z = 4-chlorobenzyloxycarbonyl
DCC = dicyclohexylcarbodiimide
DIC = diisopropylcarbodiimide
DIPEA = diisopropylethylamine
DMF = N, N-dimethylformamide
Dnp = 2,4-dinitrophenyl
Fmoc = 9-fluorenylmethoxycarbonyl
HOBt = 1-hydroxybenzotriazole Mtr = 4-methoxy-2,3,6-trimethylphenylsulfonyl
Pmc = 2,2,5,7,8-pentamethylchroman-6-sulfonyl
Trt = trityl
Mbh = 4,4'-dimethoxybenzhydryl
Tmob = 2,4,6-trimethoxybenzyl
TBTU = 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetra methyluronium tetrafluoroborate
amu = atomic mass units

General rule

The peptides were synthesized in one step with the help the standard method of solid phase peptide synthesis.

The CGRP segments were produced on the fully automatic SYRO peptide synthesizer according to the following cycle:

Splitting off of the protective group:
2 × 15 min with 25% piperidine in DMF
Washing steps:
6 × 10 min DMF
Add the pre-activated amino acid (10 equivalents, activated with TBTU, HOBt, DIPEA 1: 1: 1.5):
1 × 30 min
Washing steps:
8 × 10 min DMF.

For separation from the polymer and the side chain protective groups the peptide was treated with trifluoroacetic acid / thioanisole / thio cresol (90: 5: 5, 10 ml) stirred, concentrated in vacuo and out Diethyl ether / 0.03% HCl precipitated. After two more washes The peptide was washed with diethyl ether in an ultrasound bath in Was water dissolved and lyophilized.

The cleaning was carried out by means of reversed phase HPLC (Nucleosil C-18, 10 m, 100 A) and a gradient from 25% B to 40% in 20 min (column: 20 × 250 mm, flow: 28 ml / min, A = 0.1% aqueous  Trifluoroacetic acid and B = 0.1% trifluoroacetic acid in aceto nitrile).

The following connections were made in this way:

Claims (11)

1. From the r-CGRP-alpha-27-37 partial sequence
HF ²⁷ -V ²⁸ -P ²⁹ -T ³⁰ -N ³¹ -V ³² -G ³³ -S ³⁴ -E ³⁵ -A ³⁶ -F ³⁷ -NH ₂ (I)
derived peptides, characterized in that in formula I

• (i) one, two or three of the amino acids of positions 27 to 35 independently of one another by amino acids from the group
  K, Y, A, F, P, Hyp, S, L, Q, D, H, Acp, Pac, Tic, Hop, Aib, I and G
  are replaced or
• (ii) the amino acids of positions 27 to 34 by the sequences
  NFVPRSKISP ³⁴ , NVAPRSKISP ³⁴ , NVAPTNVGS ³⁴ or ILSSTNVGS ^{34 are} replaced or / and
• (iii) one to eight of the amino acids of positions 27 to 35 are replaced by single bonds and
• (iv) an amino acid from positions 27 to 37 can additionally be D-configured,

where the N-terminal NH ₂ groups contained in the compounds may be substituted by the radical R and R represents a protective radical or the 3,3-diphenylpropionyl, 2-propylpentanoyl, 3,5-dichlorophenoxyacetyl, 1-adamane tylacetyl -, 3,5-dibromo-4-hydroxyphenylalanyl-, 5- (4-benzhydrylpiperazin-1-yl) -1, 5-dioxo-3,3-tetramethylene-pentyl- or 5,11-dihydro-6 (6H ) -oxodibenz [b, e] azepin-11-ylcarbonyl group means,
with the exception of the compound H-YVPTNVGSEAF-NH _2,
and their salts. 2. Peptides derived from the r-CGRP-alpha-27-37 partial sequence according to claim 1, characterized in that in formula I

• (i) an amino acid of positions 27 to 35 is replaced by one of the amino acids K, Y, A, P or L) or
• (ii) F ^{27 is} replaced by Y and in addition one of the amino acids from positions 28 to 35 is replaced by A, F, Hyp, S, L, Q, D, K or H or
• (iii) F ^{27 is} replaced by Y and in addition one of the amino acids of positions 27 to 37 is D-configured or
• (iv) an amino acid from positions 27 to 35 is replaced by D and in addition a further amino acid from positions 27 to 35 is replaced by Q or P or
• (v) one amino acid of positions 27 to 35 is replaced by D and two additional adjacent amino acids of positions 27 to 35 are replaced by AH, AF, PF, Acp-F, Pac-F, Tic-F, Hyp-F, Hop -a or Aib-F are replaced or
• (vi) one or both amino acids of positions 27 and 28 are replaced by a single bond, one amino acid of positions 29 to 35 is additionally replaced by D and two further adjacent amino acids of positions 29 to 35 are replaced by AF or
• (vii) the two amino acids of positions 27 and 28 are replaced by a single bond and additionally one amino acid of positions 29 to 35 is replaced by A or two adjacent amino acids of positions 29 to 35 are replaced by AF or
• (viii) two adjacent amino acids of positions 29 to 35 are replaced by AF or PF or
• (ix) F ^{27 is} replaced by a single bond, it being possible for an amino acid from positions 30 to 35 to be replaced by P, I, L, G, Aib or D and, independently thereof, two further adjacent amino acids from positions 30 to 35 to be replaced by AF or
• (x) the amino acids of positions 27 and 28 or 27 to 29 are replaced by a single bond, it being possible for an amino acid of positions 31 to 35 to be replaced by Aib or A or two adjacent amino acids of positions 31 to 35 to be replaced by AF or
• (xi) the amino acids of positions 27 and 28 are replaced by a single bond, in addition one amino acid of positions 31 to 35 is replaced by D and two further adjacent amino acids of positions 31 to 35 are replaced by AF or
• (xii) F ^{27 is} replaced by Y and one to eight of the amino acids from positions 28 to 35 are omitted,

where the N-terminal NH ₂ groups contained in the compounds may be substituted by the radical R and R represents a protective radical or the 3,3-diphenylpropionyl, 2-propylpentanoyl, 3,5-dichlorophenoxyacetyl, 1-adamane tylacetyl -, 3,5-dibromo-4-hydroxyphenylalanyl-, 5- (4-benzhydryl-pi perazin-1-yl) -1,5-dioxo-3,3-tetramethylene-pentyl- or 5,11-dihydro-6 (6H) -oxodibenz [b, e] azepin-11-ylcarbonyl group means
with the exception of the compound H-YVPTNVGSEAF-NH ₂ ,
and their salts. 3. Peptides derived from the r-CGRP-alpha-27-37 partial sequence according to claim 1, characterized in that in formula I

• (i) an amino acid of positions 34 or 35 is replaced by K or
• (ii) F ^{27 is} replaced by Y or
• (iii) an amino acid of positions 28, 31 or 34 is replaced by P or
• (iv) an amino acid of positions 30 or 31 is replaced by D or
• (v) F ^{27 is} replaced by Y and additionally either
  one of the amino acids of positions 28 or 34 by A or
  the amino acid of position 35 by F or
  the amino acid of position 29 by Hyp or
  one of the amino acids of positions 29 to 31 by L or D or
  one of the amino acids of positions 34 or 35 is replaced by K, H, Q or L.
• (vi) the amino acid of position 31 is replaced by D and in addition a further amino acid of positions 34 or 35 by Q or P or the amino acids of positions 34 and 35 by AH, AF, PF, Acp-F, Pac-F, Tic -F, Hyp-F, Hop-F or Aib-F are replaced or
• (vii) one or both amino acids of positions 27 and 28 are replaced by a single bond, in addition the amino acid of position 31 is replaced by D and the amino acids of positions 34 and 35 are replaced by AF or
• (viii) the two amino acids of positions 27 and 28 are replaced by a single bond and additionally an amino acid of positions 34 or 35 is replaced by A or both amino acids of positions 34 and 35 are replaced by AF or
• (ix) the amino acids of positions 34 and 35 are replaced by AF or PF or
• (x) F ^{27 is} replaced by a single bond and additionally an amino acid of positions 31 or 34 by P or Aib or
• (xi) F ^{27 is} replaced by a single bond and V ²⁸ at the N-terminal amino group by a 3,3-diphenylpropionyl, 2-propylpentanoyl, 3,5-dichlorophenoxyacetyl, 1-adamantylacetyl, 3.5 -Dibromo-4-hy droxyphenylalanyl- or 5- (4-benzhydryl-piperazin-1-yl) -1,5-di oxo-3,3-tetramethylene-pentyl group is substituted, the amino acid of position 31 being substituted D and the amino acids of positions 34 and 35 can be replaced by AF or
• (xii) the amino acids of positions 27 and 28 are replaced by a single bond and P ²⁹ on the amino group by a 3,3-diphenylpropionyl-, 3,5-dichlorophen oxyacetyl-, 1-adamantylacetyl- or 5- (4-benzhydryl- pi perazin-1-yl) -1,5-dioxo-3,3-tetramethylene-pentyl group, where the amino acid of position 34 can be replaced by Aib or
• (xiii) the amino acids of positions 27 and 28 are replaced by a single bond, P ²⁹ on the amino group is substituted by a 3,5-dichlorophenoxyacetyl group, the amino acids of positions 34 and 35 are replaced by AF and additionally the amino acid of the position 31 can be replaced by D or
• (xiv) the amino acids of positions 27 to 29 are replaced by a single bond and T ³⁰ on the amino group is substituted by a 3,5-dichlorophenoxyacetyl or 1-adamane tylacetyl group,

where the N-terminal NH ₂ groups contained in the compounds can be substituted by the radical R and R represents a protective radical,
with the exception of the compound H-YVPTNVGSEAF-NH ₂ ,
and their salts. 4. The following peptides derived from the r-CGRP-alpha-27-37 partial sequence according to claim 1:
and their salts. 5. Physiologically acceptable salts of the compounds at least one of claims 1 to 4 with inorganic or organic acids or bases. 6. Medicaments containing a compound according to at least one of claims 1 to 4 or a physiologically tolerated Lich salt according to claim 5 in addition to optionally one or several inert carriers and / or diluents. 7. Use of a connection according to at least one of the An Proverbs 1 to 5 for the manufacture of a medicinal product for  acute and prophylactic treatment of headache, for Treatment of non-insulin dependent diabetes mellitus cardiovascular diseases, skin diseases, of inflammatory diseases, allergic rhinitis, from Asthma, from diseases with an excessive ge barrel enlargement and consequently reduced tissue blood circulation and morphine tolerance is. 8. Process for the manufacture of a medicament according to An Proverb 6, characterized in that by non-chemical means a connection according to at least one of claims 1 to 5 in one or more inert carriers and / or Diluent is incorporated. 9. A process for the preparation of the compounds according to claims 1 to 5, characterized in that they are prepared by means of solid phase peptide synthesis and
if necessary, a protective residue used here is split off again and / or
if desired, a compound thus obtained is converted into its salts, in particular for its pharmaceutical use into its physiologically tolerable salts. 10. Use of the compounds according to claims 1 to 5 for the production and purification of antibodies. 11. Use of the labeled compounds according to the claims 1 to 5 in RIA and ELISA assays and as diagnostic or analytical tools in neurotransmitter research.