DE3841768A1 - NEW TNF PEPTIDES - Google Patents

Abstract

New peptides of formula X-A-Gly-B-Y, in which A, B, X and Y have the meanings given in the description, are useful therapeutic agents. A process for producing them is also described.

Description

The invention relates to new tumor necrosis factor (TNF) derived Peptides, their production and their use as medicines.

By Carswell et al. (Proc. Natl. Acad. Sci. USA 72, 3666, 1975) has been reported that the serum from endotoxin-treated animals previously treated with the Mycobacteria strain Calmette-Guerin (BCG) had been infected, one caused hemorrhagic necrosis in various tumors in the mouse. This activity was attributed to the tumor necrosis factor. TNF shows also a cytostatic or cytotoxic effect on a variety of transformed cell lines in vitro, while normal human and animal cell lines are not affected (Lymphokine Reports Vol. 2, pp 235-275, Academic Press, New York, 1981). Recently the biochemical characterization and the gene described for human TNF (Nature 312, 724, 1984; J. Biol. Chem. 260, 2345, 1985; Nucl. Acids Res. 13, 6361, 1985).

The following protein structure for the mature human can be derived from these data Derive TNF:

ValArgSerSerSerArgThrProSerAspLysProValAlaHisValValAlaAsnPro
GlnAlaGluGlyGlnLeuGlnTrpLeuAsnArgArgAlaAsnAlaLeuLeuAlaAsnGly
ValGluLeuArgAspAsnGlnLeuValValProSerGluGlyLeuTyrLeuIleTyrSer
GlnValLeuPheLysGlyGlnGlyCysProSerThrHisValLeuLeuThrHisThrIle
SerArgIleAlaValSerTyrGlnThrLysValAsnLeuLeuSerAlaIleLysSerPro
CysGlnArgGluThrProGluGlyAlaGluAlaLysProTrpTyrGluProIleTyrLeu
GlyGlyValPheGlnLeuGluLysGlyAspArgLeuSerAlaGluIleAsnArgProAsp
TyrLeuAspPheAlaGluSerGlyGlnValTyrPheGlyIleIleAlaLeu

Furthermore, the TNF gene from cattle, rabbits and mice has been described (Cold Spring Harbor Symp. Quant. Biol. 51, 597, 1986).  

In addition to its cytotoxic properties, TNF is one of the main participants inflammatory reactions (Pharmac. Res. 5, 129, 1988). In the animal model TNF's involvement in septic shock (Science 229, 869, 1985) and graft versus host disease (J. Exp. Med. 166, 1280, 1987).

It has now been found that peptides with much lower Molecular weight have favorable properties.

The invention relates to peptides of the formula I

X-A-Gly-B-Y (I)

wherein

A is Asn, Asp or His,
B means Val, Met or Phe,
X for a group G-NH-CHM-CO-, G-NH-CHM-CO-W-, GR-NH-CHM-CO- or GR-NH-CHM-CO-W- and
Y represents a group -Z, -NH-CHQ-CO-Z, -V-NH-CHQ-CO-Z, -NH-CHQ-CO-UZ or -V-NH-CHQ-CO-UZ,
where in X and Y
G represents a hydrogen atom or an amino protecting group,
Z represents an OH or NH₂ group or a carboxyl protecting group or
G and Z together also mean a covalent bond or the group -CO- (CH₂) _a -NH-, where a is a number from 1 to 12,
R, U, V and W represent peptide chains from 1-4 naturally occurring α- amino acids and
M and Q are hydrogen atoms or one of the groups
-CH (CH₃) ₂, -CH (CH₃) -C₂H₅, -C₆H₅, -CH (OH) -CH₃,

(with b in the meaning of a number from 1 to 6 and T in the meaning of an OH-, CH₃O-, CH₃S-, (CH₃) ₂CH-, C₆H₅-, p-HO-C₆H₄-, HS-, H₂N-, HO -CO-, H₂N-CO-, H₂N-C (= NH) -NH group) or
M and Q together form a - (CH₂) _c -SS- (CH₂) _d -, - (CH₂) _e -CO-NH- (CH₂) _f - or - (CH₂) _e -NH-CO- (CH₂) _g - NH-CO- (CH₂) _f bridge (with c and d meaning a number from 1 to 4, e and f a number from 1 to 6 and g a number from 1 to 12),
and their salts with physiologically acceptable acids.

The peptides of formula I are made up of L-amino acids, but they can Contain 1 to 2 D-amino acids. The side chains of the trifunctional Amino acids can carry protective groups or be unprotected.

The following are particularly worth mentioning as physiologically acceptable acids: Hydrochloric acid, citric acid, tartaric acid, lactic acid, phosphoric acid, Methanesulfonic acid, acetic acid, formic acid, maleic acid, fumaric acid, Malic acid, succinic acid, malonic acid, sulfuric acid, L-glutamic acid, L-aspartic acid, pyruvic acid, mucic acid, benzoic acid, Glucuronic acid, oxalic acid, ascorbic acid, acetylglycine.

The new peptides can in particular be open-chain (G = H, amino protecting group; Z = OH, NH₂, carboxyl protecting group, M and Q not linked together), disulfide bridged (G = H, amino protecting group; Z = OH, NH₂, carboxyl protecting group; M + Q = - (CH₂) _c -SS- (CH₂) _d -), side chain bridged (G = H, amino protecting group, Z = OH, NH₂, carboxyl protecting group, M + Q = - (CH₂) _e -NH-CO- (CH₂ ) _f - or - (CH₂) _e -NH-CO- (CH₂) _g -NH-CO- (CH₂) _f -) or head-to-tail linked (G + Z = covalent bond or -CO- (CH₂) _a -NH-).

The new compounds can be made according to those known in peptide chemistry Create methods.

The peptides can be sequenced from amino acids or by fragment linking build suitable small peptides. With sequential construction the peptide chain is gradually increased by one at the C-terminus Amino acid extended. When coupling fragments, fragments different lengths are linked together, the fragments again by sequential construction from amino acids or by itself Fragment coupling can be obtained. The cyclic peptides are after synthesis of the open-chain peptides by a high dilution obtained cyclization reaction obtained.  

Both with sequential construction and with fragment coupling the building blocks are linked by forming an amide bond. For this enzymatic and chemical methods are suitable.

Chemical methods for amide bond formation are discussed in detail at Müller, Methods of Organic Chemistry Vol. XV / 2, pp 1-364, Thieme Verlag, Stuttgart, 1974; Stewart, Young, Solid Phase Peptide Synthesis, pp 31-34, 71-82, Pierce Chemical Company, Rockford, 1984; Bodanszky, Klausner, Ondetti, Peptide Synthesis, pp 85-128, John Wiley & Sons, New York, 1976 and other standard works of peptide chemistry. Especially the azide method, the symmetrical and the mixed method are preferred Anhydride method, in situ generated or preformed active esters and Formation of amide bonds with the aid of coupling reagents (activators), in particular dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), 1-ethyl-3- (3-dimethylaminopropyl) - carbodiimide hydrochloride (EDCI), n-propanephosphonic anhydride (PPA), N, N-bis (2-oxo-3-oxazolidinyl) amidophosphoric chloride (BOP-Cl), diphenylphosphoryl azide (DPPA), Castro's reagent (BOP), O-benzotriazolyl-N, N, N ′, N′-tetramethyluronium salts (HBTU), 2,5-diphenyl 2,3-dihydro-3-oxo-4-hydroxythiophene dioxide (Steglich reagent; HOTDO) and 1,1′-carbonyl-diimidazole (CDI). The coupling reagents can alone or in combination with additives such as N, N'-dimethyl-4-aminopyridine (DMAP), N-hydroxybenzotriazole (HOBt), N-hydroxybenzotriazine (HOOBt), N-hydroxysuccinimide (HOSu) or 2-hydroxypyridine can be used.

While protective groups can normally be dispensed with in enzymatic peptide synthesis, reversible protection of the reactive functional groups of the two reactants which are not involved in the formation of the amide bond is required for chemical synthesis. In chemical peptide synthesis, three protecting group techniques known from the literature are preferred: the benzyloxycarbonyl (Z), the t-butyloxycarbonyl (Boc) and the 9-fluorenylmethyloxycarbonyl (Fmoc) protective group technique. The protective group of the a -amino function of the chain-extending building block is designated in each case. The side chain protecting groups of the trifunctional amino acids are chosen so that they are not necessarily split off together with the α- amino protecting group. Müller, Methods of Organic Chemistry Vol. XV / 1, pp 20-906, Thieme Verlag, Stuttgart, 1974 provides a detailed overview of amino acid protecting groups.

The building blocks which serve to build up the peptide chains can be in solution, in suspension or by a similar method as described by Merrifield in J. Amer. Chem. Soc. 85, 2149, 1963. Particularly preferred are processes in which peptides are built up sequentially or by fragment coupling using the Z, Boc or Fmoc protective group technique, the reactants being reacted in solution, and processes in which, similar to the Merrifield technique mentioned, a reaction partner is bound to an insoluble polymeric carrier (hereinafter also called resin). The peptide is typically built up sequentially on the polymeric support using the Boc or Fmoc protective group technique, the growing peptide chain at the C-terminus being covalently linked to the insoluble resin particles (see Figs. 1 and 2). This procedure allows reagents and by-products to be removed by filtration, making recrystallization of intermediate products unnecessary.

The protected amino acids can be attached to any suitable polymer are bound, which are only insoluble in the solvents used be and a stable physical form, the light filtration enables, must have. The polymer must have a functional group contain the first protected amino acid by a covalent Binding can be firmly tied. The are suitable for this purpose various polymers, e.g. B. cellulose, polyvinyl alcohol, polymethacrylate, sulfonated polystyrene, chloromethylated copolymer of Styrene and divinylbenzene (Merrifield resin), 4-methylbenzhydrylamine resin (MBHA resin), phenylacetamidomethyl resin (Pam resin), p-benzyloxybenzyl alcohol- Resin, benzhydrylamine resin (BHA resin), 4- (hydroxymethyl) benzoyloxymethyl Harz, Harz according to Breipohl et al. (Tetrahedron Lett. 28, 565, 1987; BACHEM), HYCRAM resin (ORPEGEN) or SASRIN resin (BACHEM).

All solvents that are suitable for peptide synthesis in solution are suitable prove inert under the reaction conditions, especially water, N, N'-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetonitrile, dichloromethane (DCM), 1,4-dioxane, tetrahydrofuran (THF), N-methyl-2-pyrrolidone (NMP) and mixtures of the solvents mentioned. Peptide synthesis on the polymeric carrier in all inert organic solvents, in to which the amino acid derivatives used are soluble will; however, solvents which additionally swell with resin are preferred Have properties such as DMF, DCM, NMP, acetonitrile and DMSO, as well Mixtures of these solvents.

After successful synthesis, the peptide is removed from the polymeric carrier cleaved. The conditions under which the different types of resin split off are known from the literature. Most often find acidic and Palladium-catalyzed cleavage reactions application, especially the Cleavage in liquid anhydrous hydrogen fluoride, in anhydrous Trifluoromethanesulfonic acid, in diluted or concentrated  Trifluoroacetic acid or the palladium-catalyzed cleavage in THF or THF-DCM mixtures in the presence of a weak base such as e.g. B. Morpholine. Depending on the choice of the protective groups, these can take place under the gap conditions be preserved or also split off. Partly too Deprotection of the peptide can be useful when certain derivatization reactions or a cyclization should be carried out.

Some of the new peptides show good cytotoxic properties. A other part of the peptides has a high affinity for the cellular TNF receptor, but without having cytotoxic activity. they thus represent TNF antagonists. They bind to the natural in competition TNF to the cellular TNF receptor and thus suppress the TNF effect. The new peptides prove to be valuable medicinal products that are used for Treatment of neoplastic and autoimmune diseases as well for the control and prophylaxis of infections, inflammation and Rejection reactions can be used in transplants. By simple experiments can be clarified which mode of action the own individual peptides. With a TNF-sensitive cell Cytotoxicity of the peptide by incubation of the cell line in the presence of the Peptide determined. In a second experiment you incubate the Cell line with the corresponding peptide in the presence of a lethal agent TNF amount. This can demonstrate the TNF-antogonizing effect will. In addition, the affinity is determined by an in vitro binding experiment of the peptide to the cellular TNF receptor.

The biological characterization of the new peptides on their agonistic or antagonistic effect took place in the following test systems:

I. cytotoxicity test on TNF-sensitive indicator cells,
II. Competition cytotoxicity test on TNF-sensitive indicator cells,
III. Competition receptor binding test on TNF receptor expressing indicator cells.

I. Cytotoxicity test

The agonistic evaluation of the new peptides is based on their cytotoxic effect on TNF-sensitive cells (e.g. L929, MCF-7, A204, U937). The L929 and MCF-7 test was performed as follows:  

• 1. 100 ul culture medium with 3 to 5 × 10³ freshly trypsinized itself L929 cells (mouse) in exponential growth or MCF-7 cells (human) were placed in the well Pipetted 96-well flat-bottom culture plate. The plate was over Incubated overnight at 37 ° C in the incubator. The one with steam saturated air in the incubator contained 5 vol% CO₂.
• The L929 culture medium contained 500 ml MEM Earle 1 × (Boehringer, Mannheim), 50 ml heat-inactivated (30 min, 56 ° C) fetal Calf serum (FCS), 50 ml L-glutamine (200 mM), 5 ml 100 × non-essential amino acid, 3 ml 1M Hepes buffer pH 7.2 and 50 ml gentamycin (50 mg / ml).
• The MCF-7 culture medium contained 500 ml MEM Dulbecco 1x (Boehringer, Mannheim), 100 ml heat-inactivated (30 min, 56 ° C) FCS, 5 ml L-glutamine and 5 ml 100 × non-essential amino acids.
• 2. The following day, 100 ul of the peptide solution to be tested were added given to the cell cultures and titrated serially twice. In addition some cell controls (i.e., not with peptide dilution treated cell cultures) and some rhu-TNF controls (i.e., with recombinant human TNF-treated cell cultures). The culture plate was exposed for 48 hours at 37 ° C in one atmosphere Steam-saturated air incubated with 5 vol.% CO₂.
• 3. The percentage of surviving cells in those with peptide dilution treated cultures was by means of crystal violet staining certainly. To do this, the liquids from the wells removed by knocking off the test plate. In every well 50 ul crystal violet solutions were pipetted.
• The crystal violet solution had the following composition: 3.75 g crystal violet
  1.75 g NaCl
  161.5 ml of ethanol
  43.2 ml 37% formaldehyde
  ad 500 ml water
• The crystal violet solution remained in the wells and for 20 min was then also knocked off. Then the Plates washed 5 times each by immersing them in water  to remove the non-cell-bound dye. The cell-bound Dye was removed by adding 100 ul reagent solution (50% ethanol, 0.1% glacial acetic acid, 49.9% water) in each well extracted from the cells.
• 4. By shaking the plates for 5 min each was obtained Deepen an evenly colored solution. To determine the The extinction of the staining solution in the surviving cells individual wells measured at 540 nm.
• 5. Then, based on the cell control, the 50% cytotoxicity value defined and the reciprocal of the sample dilution that leads to 50% cytotoxicity as the cytotoxic activity of the investigated sample determined.

II. Competition cytotoxicity test

The antagonistic evaluation of the peptides is based on their Property, the cytotoxic effect of rhu-TNF on TNF-sensitive Compete cells (e.g. L929, MCF-7, A204, U937). The Competition cytotoxicity test with L929 and MCF-7 cells was as follows carried out:

• 1. 100 ul culture medium with 3 to 5 × 10³ freshly trypsinized itself L929 cells (mouse) in exponential growth or MCF-7 cells (human) were placed in the well Pipetted 96-well flat-bottom culture plate. The plate was over Incubated overnight at 37 ° C in the incubator. The one with steam saturated air in the incubator contained 5 vol% CO₂.
• The L929 culture medium contained 500 ml MEM Earle 1 × (Boehringer, Mannheim), 50 ml for 30 min at 56 ° C heat-inactivated FCS, 5 ml L-glutamine (200 mM), 5 ml 100 × non-essential amino acid, 3 ml 1M Hepes buffer pH 7.2 and 500 µl gentamycin (50 mg / ml).
• The MCF-7 culture medium contained 500 ml MEM Dulbecco 1x (Boehringer, Mannheim), 100 ml heat-inactivated (30 min, 56 ° C) FCS, 5 ml L-glutamine (200 mM) and 5 ml 100 × non-essential Amino acids.
• 2. The following day, 100 ul of the peptide solution to be tested were added given to the cell cultures and titrated serially twice. To this Cell cultures were then 100 ul of a rhu-TNF dilution in Culture medium in the final concentration in the cell culture  Has 80-100% cytotoxic effect. In addition, some Cell controls (i.e., not with peptide solution and not with rhu-TNF solution treated cell cultures) and some rhu-TNF controls (= only treated with rhu-TNF solution Cell cultures). The culture plate was then at 48 hours 37 ° C in an atmosphere of water vapor-saturated air 5 vol.% CO₂ incubated.
• 3. The percentage of surviving cells in the solution solution treated cultures was by means of crystal violet staining certainly. To do this, the liquids from the wells removed by knocking off the test plate. In every well 50 ul crystal violet solutions were pipetted.
• The crystal violet solution had the one given in II.3 Composition.
• The crystal violet solution remained in the wells and for 20 min was then also knocked off. Then the Plates washed 5 times each by immersing them in water to remove the non-cell-bound dye. The cell-bound dye was added by adding 100 ul Reagent solution (50% ethanol, 0.1% glacial acetic acid, 49.9% water) in extracted each well from the cells.
• 4. By shaking the plates for 5 min each was obtained Deepen an evenly colored solution. To determine the The extinction of the staining solution in the surviving cells individual wells measured at 540 nm.
• 5. Then, based on the cell control and the rhu-TNF control defined the 50% competition value and the Sample concentration at the submitted rhu-TNF concentration leads to 50% competition of rhu-TNF cytotoxicity, as determine the antagonistic activity of the examined sample.

III. Competition receptor binding test

Both the agonistic and the antagonistic effects of Peptides require that the latter bind to the TNF receptor. The means that peptides with agonistic or antagonistic effect and rhu-TNF to bind to the TNF receptor on TNF-sensitive Indicator cells (e.g. U937) compete. The competition receptor binding test was carried out as follows:  

• 1. 100 µl medium with different concentrations of the test sample The peptide and the rhu-TNF (= control) were placed in the reaction vessels pipetted. The medium contained 500 ml PBS (Boehringer, Mannheim), 10 ml heat-inactivated (30 min, 56 ° C) FCS and 100 mg Sodium azide.
• 2. 100 µl medium was then labeled with 1 ng ¹²⁵iodine rhu-TNF (lactoperoxidase method according to Bolton) into the reaction vessels given and mixed. To determine the non-specific Binding (NSB) became the ¹²⁵iodine-labeled in the reaction vessels rhu-TNF (1 ng ¹²⁵J-rhu-TNF in 100 µl medium) with 200 times Excess of non-radioactively labeled rhu-TNF (200 ng rhu-TNF mixed in 100 ul medium).
• 3. Then 100 ul medium with 2 × 10⁶ U937 cells (human) were in the Pipette and mix reaction tubes. The reaction vessels (Test volume 300 µl) were incubated at 0 ° C for 90 min. After 45 min the reaction batches were mixed again.
• 4. After the incubation period, the cells were 5 min at 1800 rpm and Centrifuged at 4 ° C, washed 3 times with medium, quantitatively in Transferred tubes and the cell-bound radioactivity in a Clini Gamma Counter 1272 (LKB Wallac).
• 5. After correcting the measured values for the specific binding, based on the total binding, the 50% competition value defined and the sample concentration that is presented at the ¹²⁵J-rhu-TNF concentration to 50% competition of ¹²⁵J-rhu-TNF binding results as a competitive activity of determine the examined sample.

The following examples are intended to explain the invention in more detail. The proteogenic amino acids are abbreviated in the examples with the well-known three-letter code. In addition: Aad = α- aminoadipic acid, Ac = acetic acid, Aoc = 8-aminooctanoic acid, Ape = 5-aminopentanoic acid, Hcy = homocysteine, Hly = homolysin, Orn = ornithine, Dap = 2,3-diaminopropionic acid.

A. General labor regulations I. The synthesis of the peptides according to claim 1 was carried out with the aid of Standard methods of solid phase peptide synthesis on a fully automated Model 430A peptide synthesizer from APPLIED BIOSYSTEMS. The device uses different for the Boc and Fmoc protection group technology Synthesis cycles

a) Synthesis cycle for Boc protection group technology

b) Synthesis cycle for Fmoc protection group technology

II. Processing of the peptide resins obtained according to Ia

The peptide resin obtained according to Ia was dried in vacuo and in a Reaction vessel of a Teflon HF apparatus (from PENINSULA) transferred. After adding a scavenger, preferably anisole (1 ml / g resin), as well in the case of tryptophan-containing peptides of a thiol for removal the indolene formyl group, preferably ethanedithiol (0.5 ml / g Resin) was condensed with cooling with liquid N₂ hydrogen fluoride (10 ml / g resin). The mixture was allowed to warm to 0 ° C and stirred at this temperature for 45 min. Then the hydrogen fluoride stripped off in vacuo and the residue washed with ethyl acetate, to remove the rest of the scavengers. The peptide was made with 30% acetic acid extracted, filtered and the filtrate lyophilized.

To produce peptide hydrazines, the peptide resin (Pam or Merrifield resin) suspended in DMF (15 ml / g resin) and after transfer stirred with hydrazine hydrate (20 equivalents) for 2 days at room temperature. For working up, the resin was filtered off and the filtrate Evaporated dry. The residue was made from DMF / Et₂O or MeOH / Et₂O crystallized.

III. Processing of the peptide resins obtained according to Ib

The peptide resin obtained according to Ib was dried in vacuo and then depending on the amino acid composition of a subjected to the following splitting procedures (Wade, Tregear, Howard Florey Fmoc-Workshop Manual, Melbourne 1985).  

The suspension of the peptide resin in the appropriate TFA mixture was stirred at room temperature for the specified time, after which the The resin is filtered off and washed with TFA and DCM. The filtrate and the Wash solutions were largely concentrated and the peptide by addition precipitated from diethyl ether. After cooling in the ice bath, the Filtered precipitate, taken up in 30% acetic acid and lyophilized.

IV. Purification and characterization of the peptides

The purification was carried out by means of gel chromatography (SEPHADEX G-10, G-15/10% HOAc; SEPHADEX LH20 / MeOH) and subsequent medium pressure chromatography (Stationary phase: HD-SIL C-18, 20-45 µ, 100 Å; mobile phase: gradient with A = 0.1% TFA / MeOH, B = 0.1% TFA / H₂O).  

The purity of the end products obtained was determined using analytical HPLC (Stationary phase: 100 × 2.1 mm VYDAC C-18, 5 µ, 300 Å; mobile phase = CH₃CN / H₂O gradient, buffered with 0.1% TFA, 40 ° C) determined. To Amino acid analysis and fast atom bombardment were characterized. Mass Spectroscopy.

B. Special working regulations Example 1 H-Ala-Asn-Gly-Val-Glu-NH₂

1.28 g Boc-Glu (OBzl) -MBHA resin (substitution∼0.39 mmol / g), accordingly a batch size of 0.5 mmol, were according to AIa with 2 mmol

Boc-Val-OH
Boc-Gly-OH
Boc-Asn-OH
Boc-Ala-OH

implemented.

After the synthesis had ended, the peptide resin was deprotected at the N-terminal (Execution of steps 1-3 according to AIa) and then in a vacuum dried; the yield was 1.35 g.

0.7 g of the resin thus obtained was subjected to HF cleavage in accordance with AII subject. The crude product (81 mg) was purified by gel filtration (SEPHADEX G-10) and medium pressure chromatography (cf. AIV; 40-60% A; 0.25% min⁻¹) cleaned.

Example 2 Ac-Leu-Ala-Asn-Gly-Val-Glu-OH

0.46 g Fmoc-Glu (OtBu) -p-alkoxybenzyl alcohol resin (Substitution∼0.55 mmol / g), corresponding to a batch size of 0.25 mmol, were according to AIb with 1 mmol

Fmoc-Val-OH
Fmoc-Gly-OH
Fmoc-Asn-OH
Fmoc-Ala-OH
Fmoc-Leu-OH

implemented.  

After the synthesis was complete, the N-terminus was acetylated (execution of the Steps 2-4 and 8-9 according to AIb). The peptide resin obtained was in Vacuum dried; the yield was 0.54 g.

The crude peptide (129 mg) obtained after the TFA cleavage according to AIII was by gel filtration (SEPHADEX G-10) and medium pressure chromatography (cf. AIV; 40-60%; 0.25% min⁻¹) cleaned. There were 87 mg of pure product receive.

Analogously to Examples 1 and 2, the following can be produced:

3. H-Ala-Asn-Gly-Val-Glu-OH
4. Ac-Ala-Asn-Gly-Val-Glu-OH
5. Ac-Ala-Asn-Gly-Val-Glu-NH₂
6. H-Leu-Ala-Asn-Gly-Val-Glu-OH
7. H-Leu-Ala-Asn-Gly-Val-Glu-NH₂
8. Ac-Leu-Ala-Asn-Gly-Val-Glu-NH₂
9. H-Leu-Ala-Asn-Gly-Val-Glu-Leu-OH
10. Ac-Leu-Ala-Asn-Gly-Val-Glu-Leu-OH
11. H-Leu-Ala-Asn-Gly-Val-Glu-Leu-NH₂
12. Ac-Leu-Ala-Asn-Gly-Val-Glu-Leu-NH₂
13. H-Leu-Leu-Ala-Asn-Gly-Val-Glu-Leu-Arg-OH
14. Ac-Leu-Leu-Ala-Asn-Gly-Val-Glu-Leu-Arg-OH
15. H-Leu-Leu-Ala-Asn-Gly-Val-Glu-Leu-Arg-NH₂
16. Ac-Leu-Leu-Ala-Asn-Gly-Val-Glu-Leu-Arg-NH₂
17. H-Ala-Leu-Leu-Ala-Asn-Gly-Val-Glu-Leu-Arg-OH
18. Ac-Ala-Leu-Leu-Ala-Asn-Gly-Val-Glu-Leu-Arg-NH₂
19. H-Leu-Ala-Asn-Gly-Phe-Glu-OH
20. Ac-Leu-Ala-Asn-Gly-Phe-Glu-NH₂
21. Ac-Leu-Ala-Asn-Gly-Met-Glu-Leu-NH₂
22. Ac-Leu-Leu-Ala-His-Gly-Val-Glu-Leu-Arg-NH₂
23. Ac-Ile-Ala-Asn-Gly-Val-Glu-NH₂

Example 24

0.98 g Boc-Cys (pMB) -MBHA resin (substitution ∼0.51 mmol / g), accordingly a batch size of 0.5 mmol according to AIa with 2 mmol

Boc-Glu (OBzl) -OH
Boc-Val-OH
Boc-Gly-OH
Boc-Asn-OH
Boc-Cys (pMB) -OH

implemented.  

After the synthesis was complete, the N-terminus was acetylated (execution of the Steps 1-6 and 14-16 according to AIa).

The peptide resin obtained was dried in vacuo; the yield was 1.3 g.

0.65 g of the resin thus obtained was subjected to HF cleavage in accordance with AII subject. The lyophilized crude product became 0.1% in 2 l Acetic acid added and the pH then with aqueous ammonia 8.4 set. 0.01 N was slowly added under an argon atmosphere K₃ [Fe (CN) ₆] solution added dropwise until the yellowish green color is longer than 15 minutes remained. Another 1 was stirred, then with glacial acetic acid acidified to pH 4.5 and with 15 ml of an aqueous suspension Anion exchanger (BIORAD 3 × 4A, chloride form) added. After 30 min the ion exchange resin was filtered off, the filtrate on a rotary evaporator concentrated to 100 ml and then lyophilized.

All solvents used were previously saturated with nitrogen to prevent possible oxidation of the free cysteine residues.

The crude product was purified by gel chromatography (SEPHADEX G-15) and medium pressure chromatography (cf. AIV; 55-75% A; 0.25% min⁻¹) cleaned. It 36 mg of pure product were obtained.

Analogously to Example 24, the following can be produced:

Example 71

1.22 g Boc-Lys (Z) -MBHA resin (substitution ∼0.41 mmol / g), accordingly a batch size of 0.5 mmol, were according to AIa with 2 mmol

Boc-Val-OH
Boc-Gly-OH
Boc-Asn-OH
Boc-Ala-OH
Boc-Glu (OBzl) -OH

implemented. After the synthesis was complete, the N-terminus was acetylated (Execution of steps 1-6 and 14-16 according to AIa). The received Peptide resin was dried in vacuo; the yield was 1.47 g.

The crude product (274 mg) obtained after the HF cleavage according to AII was in 500 ml degassed DMF dissolved, with 210 mg NaHCO₃ and 0.12 ml diphenylphosphoryl azide transferred. The mixture was 3 days at room temperature stirred and then evaporated to dryness. The crude peptide was purified by gel chromatography (SEPHADEX LH 20). It became 104 mg Get pure product.

Example 72

1 g of resin according to Breipohl et al. (BACHEM), according to a batch size of 0.5 mmol was according to AIb with 2 mmol

Fmoc-Lys (Boc) -OH
Fmoc-Glu (OBzl) -OH
Fmoc-Val-OH
Fmoc-Gly-OH
Fmoc-Asn-OH
Fmoc-Ala-OH
Fmoc-Glu (OtBu) -OH

implemented. After the synthesis was complete, the N-terminus was acetylated (Execution of steps 2-4 and 8-9 according to AIb). The peptide resin was dried in vacuo; Yield 1.42 g.

The crude product obtained after the TFA cleavage according to AIII (286 mg) was dissolved in 500 ml degassed DMF. After adding 210 mg of NaHCO₃ and 0.12 ml Diphenylphosphoryl azide was stirred for 3 days at room temperature. Then was evaporated to dryness, and the crude peptide by gel chromatography (SEPHADEX LH 20) cleaned. The isolated monomer (175 mg) was dissolved in 10 ml MeOH dissolved and after addition of 20 mg Pd / C (10%) 8 h at normal pressure hydrated. The one obtained after filtering off the catalyst and evaporating Product was by medium pressure chromatography (cf. AIV; 55-75% A; 0.25% min⁻¹) cleaned. 108 mg of pure product were obtained.

Analogously to Examples 71 and 72, the following can be produced:

Example 95

1.19 g Fmoc-Leu-p-alkoxybenzyl alcohol resin (substitution ∼0.42 mmol / g), corresponding to a batch size of 0.5 mmol, was according to AIb with 2 mmol

Fmoc-Glu (OBzl) -OH
Fmoc-Val-OH
Fmoc-Gly-OH
Fmoc-Asn-OH
Fmoc-Ala-OH

implemented. When synthesis was complete, the peptide resin became N-terminal deprotected (execution of steps 2-4 according to AIb) and then in Vacuum dried. The yield was 1.35 g.

The crude peptide obtained after TFA cleavage according to AIII was in 500 ml degassed DMF solved. After adding 210 mg of NaHCO₃ and 0.12 ml Diphenylphosphoryl azide was stirred for 3 days at room temperature. Then was evaporated to dryness, and the crude peptide by gel chromatography (SEPHADEX LH 20) cleaned. The isolated monomer (106 mg) was dissolved in 10 ml MeOH dissolved and after addition of 20 mg Pd / C (10%) 8 h at normal pressure  hydrated. The one obtained after filtering off the catalyst and evaporating Product was by medium pressure chromatography (cf. AIV; 55-75% A; 0.25% min⁻¹) cleaned. 75 mg of pure product were obtained.

The following can be produced analogously to Example 95:

Claims (8)

1. The invention relates to peptides of the formula I, XA-Gly-BY (I) in which A is Asn, Asp or His,
B means Val, Met or Phe,
X for a group G-NH-CHM-CO-, G-NH-CHM-CO-W-, GR-NH-CHM-CO- or GR-NH-CHM-CO-W- and
Y represents a group -Z, -NH-CHQ-CO-Z, -V-NH-CHQ-CO-Z, -NH-CHQ-CO-UZ or -V-NH-CHQ-CO-UZ,
where in X and Y
G represents a hydrogen atom or an amino protecting group,
Z represents an OH or NH₂ group or a carboxyl protecting group or
G and Z together also mean a covalent bond or the group -CO- (CH₂) _a -NH-, where a is a number from 1 to 12,
R, U, V and W represent peptide chains from 1-4 naturally occurring α- amino acids and
M and Q are hydrogen atoms or one of the groups
-CH (CH₃) ₂, -CH (CH₃) -C₂H₅, -C₆H₅, -CH (OH) -CH₃, (with b in the meaning of a number from 1 to 6 and T in the meaning of an OH-, CH₃O-, CH₃S-, (CH₃) ₂CH-, C₆H₅-, p-HO-C₆H₄-, HS-, H₂N-, HO -CO-, H₂N-CO-, H₂N-C (= NH) -NH group) or
M and Q together form a - (CH₂) _c -SS- (CH₂) _d -, - (CH₂) _e -CO-NH- (CH₂) _f - or - (CH₂) _e -NH-CO- (CH₂) _g - NH-CO- (CH₂) _f bridge (with c and d meaning a number from 1 to 4, e and f a number from 1 to 6 and g a number from 1 to 12),
and their salts with physiologically acceptable acids. 2. Peptides according to claim 1, wherein G is a hydrogen atom or an amino protecting group and Z represents a hydroxy or amino group or a carboxyl protecting group represent and M and Q are not connected. 3. Peptides according to claim 1, wherein G represents a hydrogen atom or an amino protecting group and Z represents a hydroxyl or amino group or a carboxyl protecting group and M and Q together represent a - (CH₂) _c -SS- (CH₂) _d bridge. 4. Peptides according to claim 1, wherein G represents a hydrogen atom or an amino protecting group and Z represents a hydroxy or amino group or a carboxyl protecting group and M and Q together form a group - (CH₂) _e -NH-CO- (CH₂) _f - or - ( CH₂) _e -NH-CO- (CH₂) _g -NH-CO- (CH₂) _f mean. 5. Peptides according to claim 1, wherein G + Z together represent a covalent bond or -CO- (CH₂) _a -NH-. 6. Peptides according to claim 1 to 5 for use in combating Diseases. 7. Use of the peptides according to claims 1 to 5 for combating neoplastic diseases and autoimmune diseases as well as Control and prophylaxis of infections, inflammation and Rejection reactions in transplants. 8. A method for producing the peptides according to claim 1 to 5, characterized characterized in that these are known in peptide chemistry Produces methods.