DE4041187A1 - NEW TNF PEPTIDES - Google Patents

Abstract

The description relates to peptides of formula (I), in which A, B, D, E, F, J, T, X, Y and Z have the significances given in the specification, and their production. The novel peptides are suitable for the treatment of diseases.

Description

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

From Carswell et al. (Proc Natl Acad Sci USA 72, 3666, 1975) has been reported that the serum of endotoxin-treated animals, previously with the Mycobacteria strain Calmette-Guerin (BCG) had been infected, one haemorrhagic necrosis in various tumors in the mouse. This activity was attributed to the tumor necrosis factor. TNF shows also a cytostatic or cytotoxic effect against 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 for human TNF (Nature 312, 724, 1984; J. Biol. Chem. 260, 2345, 1985; Nucl. Acids Res. 13, 6361, 1985).

From this data, the following protein structure can be found for the mature human Derive TNF:

ValArgSerSerSerArgThrProSerAspLysProValAlaHisValValAlaAsnPro
GlnAlaGluGlyGlnLeuGlnTrpLeuAsnArgArgAlaAsnAlaLeuLeuAlaAsnGly
ValGluLeuArgAspAsnGlnLeuValValProSerGluGlyLeuTyrLeuIleTyrSer
GlnValLeuPheLysGlyGlnGlyCysProSerThrHisValLeuLeuThrHisThrIle
SerArgIleAlaValSerTyrGlnThrLysValAsnLeuLeuSerAlaIleLysSerPro
CysGlnArgGluThrProGluGlyAlaGluAlaLysProTrpTyrGluProIleTyrLeu
GlyGlyValPheGlnLeuGluLysGlyAspArgLeuSerAlaGluIleAsnArgProAsp
TyrLeuAspPheAlaGluSerGlyGlnValTyrPheGlyIleIleAlaLeu

Furthermore, the TNF gene of bovine, rabbit and mouse has been described (Cold Spring Harbor Symp. Quant. Biol. 51, 597, 1986).  

In addition to its cytotoxic properties, TNF is one of the main players in inflammatory reactions (Pharmac. Res., 5, 1129, 1988). In the animal model the involvement of TNF 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 significantly lower molecular weight have favorable properties.

The invention relates to compounds of the formula I.

wherein
A, B, D, E and J are hydrogen atoms or one of the groups

(with b in the meaning of a number from 1 to 6 and T in the meaning of an OH, CH₃S-, (CH₃) ₂CH-, C₆H₅-, p-HO-C₆H₄-, H₂N-, HS-, HO-CO- , H₂N-CO or H₂N-C (= NH) -NH group)
F Asp, Asn or His means
T¹-T⁶ are hydrogen atoms or methyl groups,
Z¹-Z⁶ each represent an oxygen atom or two hydrogen atoms
X represents the groups G-, G-NH-CHM-CO-, G-NH-CHM-CO-W-, GR-NH-CHM-CO- or GR-NH-CHM-CO-W- and
-P, -NH-CHQ-CO-P, -V-NH-CHQ-CO-P, -NH-CHQ-CO-UP, -V-NH-CHQ-CO-UP,
-NH-CHQ-K-NH-CHL-CO-P, -V-NH-CHQ-K-NH-CHL-CO-P,
Is -NH-CHQ-K-NH-CHL-CO-UP or -V-NH-CHQ-K-NH-CHL-CO-UP,
where in X and Y
G represents a hydrogen atom or an amino-protecting group,
P is an OH or NH₂ group or a carboxyl protecting group or
G and P together also represent a covalent bond or the group -CO- (CH₂) _a -NH-, where a is a number from 1 to 12,
K, R, U, V and W are peptide chains of 1-5 naturally occurring α-amino acids,
L, M and Q are hydrogen atoms or one of the groups
-CH₃, -CH₂-CH (CH₃) ₂, -CH₂-CO-NH₂, - (CH₂) ₃-NH-C (NH) -NH₂, - (CH₂) ₂-CO-NH₂,
-CH (CH₃) ₂, -CH₂OH, - (CH₂) ₂COOH, or
A, B, D, M, L and Q in pairs one or two - (CH₂) _c -SS- (CH₂) _d -
- (CH₂) _e -CO-NH- (CH₂) _f - or - (CH₂) _e -NH-CO- (CH₂) _g -NH-CO- (CH₂) _f -Bridge (s)
(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 the formula I are composed of L-amino acids, but they can Contain 1 to 2 D-amino acids or N-methyl-amino acids. The side chains the trifunctional amino acids can carry protecting groups or unprotected.

Particularly suitable physiologically acceptable acids are: 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 be especially open-chain
(G = H, amino-protecting group, P = OH, NH₂, carboxyl-protecting group, A, B, D, M, L, Q not connected to each other),
Disulfide bridged (G = H, amino protecting group, P = OH, NH₂, carboxyl protecting group
A + Q, B + Q, D + Q or M + Q = - (CH₂) _c -SS- (CH₂) _d -),
Side-chain bridged (G = H, amino-protecting group, P = OH, NH₂, carboxyl-protecting group, A + Q, B + Q, D + Q
or M + Q = - (CH₂) _e -NH-CO- (CH₂) _f - or - (CH₂) _e -NH-CO- (CH₂) _g -NH-CO- (CH₂) _f -),
Head-to-tail linked (G + P = covalent bond
or -CO- (CH₂) _a -NH-) OH, NH₂, carboxyl-protecting group) or bicyclic (G = H, amino-protecting group;
P = OH, NH₂, carboxyl protecting group, M + D and A + Q or A + L and D + Q
correspond respectively to - (CH₂) _c -SS- (CH₂) _d bridges or - (CH₂) _e -NH-CO- (CH₂) _f bridges)
or contain reduced peptide bonds.

The new compounds can be known in peptide chemistry Establish methods.

Thus, the peptides can be sequenced from amino acids or by fragment linkage build up suitable small peptides. In the sequential structure The peptide chain, starting at the C-terminus, becomes one step at a time Extended amino acid. In the fragment coupling, fragments of different Length be linked together, with the fragments again by sequential structure of amino acids or by their turn Fragment coupling can be obtained. The cyclic peptides become after synthesis of the open-chain peptides by high dilution obtained cyclization reaction.

Both the sequential structure, as well as the fragment coupling must the building blocks are linked by formation of an amide formation. For this Enzymatic and chemical methods are suitable.

Chemical methods for amide bond formation are discussed in detail Müller, Methods of Organic Chemistry Vol XV / 2, pp 1-364, Thieme Publisher, 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 preferred are the azide method, the symmetric and mixed anhydride method, in situ generated or preformed active esters and amide bond formation 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 acid chloride (BOP-Cl), diphenyl phosphoryl azide (DPPA), Castro's reagent (BOP), O-benzotriazolyl-N, N, N ', N'- tetramethyluronium salts (TBTU), 2,5-diphenyl-2,3-dihydro-3-oxo-4-hydroxy  thiophene dioxide (Steglich's reagent, HOTDO), bromo-tris-pyrrolidino- phosphonium hexafluorophosphate (PyBroP) and 1,1'-carbonyldiimidazole (CDI). The coupling reagents may be used 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 in enzymatic peptide synthesis normally protective groups can be dispensed with, is for chemical synthesis reversible protection of those not involved in the formation of the amide bond reactive functional groups of the two reactants required. In the chemical peptide syntheses are three known from the literature protection Group calculations preferably: The benzyloxycarbonyl (Z) -, the t-butyloxycarbonyl (Boc) - and the 9-fluorenylmethyloxycarbonyl (Fmoc) protection group technique. In each case, the protective group of the α-amino function of the chain-extending building block. The side chain protecting groups of trifunctional Amino acids are chosen so that they are not necessarily be cleaved together with the α-amino protecting group. A detailed Overview of amino acid protecting groups are Müller, methods of Organic Chemistry Vol XV / 1, pp 20-906, Thieme Verlag, Stuttgart, 1974.

The building blocks that serve to build the peptide chain can be dissolved in solution, in Suspension or by a similar method as described by Merrifield in J. Amer. Chem. Soc. 85, 2149, 1963, reacted become. Particularly preferred are methods in which peptides are sequential or by fragment coupling using the Z, Boc or Fmoc Protective group technology are constructed, the reaction partners in Solution to be reacted, as well as methods in which, similar the aforementioned Merrifield technique, a reaction partner to an insoluble polymeric carrier (hereinafter also called resin) bound to Reaction is brought. The peptide is typically used Boc or Fmoc protective group technique sequentially on the polymeric Carrier constructed with the growing peptide chain at the C-terminus covalent is associated with the insoluble resin particles (see Fig. 1 and 2). These Operation allows reagents and by-products by filtration to remove, the recrystallization of intermediates is thus unnecessary.

The protected amino acids can be added to any suitable polymers which are insoluble only in the solvents used and a stable physical form, easy filtration allows to have. The polymer must be a functional group contain, to which the first protected amino acid by a covalent Binding can be tied tightly. For this purpose are the  various polymers, eg. Cellulose, polyvinyl alcohol, polymethacrylate, sulfonated polystyrene, chloromethylated copolymer of Styrene and divinylbenzene (Merrifield resin), 4-methylbenzhydrylamine resin (MBHA resin), phenyl acetamidomethyl resin (Pam resin), p-benzyloxybenzyl alcohol Resin, benzhydrylamine resin (BHA resin), 4- (hydroxymethyl) benzoyl oxymethyl resin, resin according to Breipohl et al. (Tetrahedron Lett. 28, 565, , 1987; BACHEM), HYCRAM resin (ORPEGEN), SASRIN resin (BACHEM) (Dimethoxyphenylaminomethyl) phenoxy resin, RINK resin; (Tetrahedron Lett.) 28, 3787 (1987, Novabiochem) or o-chloro-trityl resin (BIOHELLAS).

For the peptide synthesis in solution, all solvents are suitable prove to be 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. The peptide synthesis The polymeric carrier can be dissolved in all inert organic solvents, in where the amino acid derivatives used are soluble become; However, preferred are solvents which additionally resin-swelling Possess properties such as DMF, DCM, NMP, acetonitrile and DMSO, as well as Mixtures of these solvents

After successful synthesis, the peptide is cleaved from the polymeric support. The conditions under which the different resin types can be split off, are known from the literature. Most commonly find acid and Palladium-catalyzed cleavage reactions application, in particular the Cleavage in liquid anhydrous hydrogen fluoride, in anhydrous Trifluoromethanesulfonic acid, in dilute or concentrated trifluoroacetic acid or the palladium-catalyzed cleavage in THF or THF-DCM- Mixtures in the presence of a weak base such. B. morpholine. Depending on Choice of protecting groups can be obtained under the cleavage conditions stay or be split off as well. Also a partial deprotection of the peptide may be useful if certain derivatization reactions or a cyclization should be carried out.

Part of the new peptides shows TNF activity. These pose TNF agonists Another part of the peptides has a high affinity for the cellular TNF receptor but without TNF-like activity to own. They represent TNF antagonists. They bind in competition to natural TNF to the cellular TNF receptor, thus suppressing the TNF effect. The new peptides prove to be valuable drugs, for the treatment of neoplastic diseases and autoimmune diseases as well as to control and prophylaxis of infections, inflammations and rejection reactions in transplants can.  

The characterization of the new peptides on their agonistic or Antagonistic action was essentially in the following test systems:

• I. Receptor binding test
• II. Monocyte Activation Test: Induction of TNF mRNA
• III. Granulocyte activation test: release of oxygen radicals
• IV. Cytotoxicity test

ad I. Receptor binding test

Both the agonistic and antagonistic effects of peptides require that the latter bind to the TNF receptor. This means that peptides with agonistic or antagonistic action and rHu-TNF compete for binding at the TNF receptor on TNF-sensitive indicator cells. A competition receptor binding test (Int. J. Cancer 38, 127 (1986) was performed as follows:
Using human U937 cells (2 × 10⁶ cells per group, 300 μl assay volume) and 1 ng ¹²⁵I-labeled rHuTNF (bolton lactoperoxidase method), the peptide concentration was determined which resulted in a 50% displacement of the submitted125 I -rHuTNF leads from the TNF receptor.

ad II. Monocyte activation test

The agonistic evaluation of the new peptides is based on their TNFmRNA-inducing activity in human monocytes. The test (J. Immunol., 144, 1144 (1990) was performed as follows:
Human peripheral blood monocytes (5 to 10 x 10⁶ per group, 3 ml assay volume) were incubated with the peptides for 2 hours at 37 ° C. RNA was extracted and induction of TNF mRNA was measured by Northern blot analysis.

The antagonistic evaluation of the new peptides is based on their ability to in this test system the rHuTNF-induced induction of TNFmRNA too inhibit.

ad III: granulocyte activation test

The agonistic activity of the new peptides is based on their induction of oxygen-oxygen release in human granulocytes. The test (Methods in Enzymology 133, 449 (1986) was carried out as follows:
Using polymorphonuclear neutrophils (2.5 x 10⁵ per group, 500 μl assay volume), the peptide-induced release of radical oxygen was measured in a chemiluminescent assay.

The antagonistic evaluation of the new peptides is based on their property, in this test system the rHuTNF-induced radical oxygen Inhibit release.

ad IV: cytotoxicity test

The agonistic evaluation of the new peptides is based on their cytotoxic effect on TNF-sensitive tumor cells. The test was carried out as follows:
Using human MCF-7 cells (2 x 10⁴ cells per group, 200 ul assay volume), the peptide concentration was determined in a proliferation test, which after incubation for 24 hours, the incorporation of ³H-TdR in the DNA of MCF-7 cells to 50% inhibits.

The antagonistic evaluation of the new peptides is based on their ability to inhibit the cytotoxic effects of rHuTNF in this test system.

The following examples are intended to explain the invention in more detail. The proteogenic amino acids are in the examples with the well-known three-letter Code abbreviated. In addition, Ac = acetic acid, Sar = sarcosine.

A. General working instructions

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 Peptide Synthesizer Model 430A from APPLIED BIOSYSTEMS. The device uses different Boc and Fmoc protection group technology Synthetic cycles.

a) Synthesis cycle for the Boc protective group technique 1. 30% trifluoroacetic acid in DCM | 1 × 3 min 2. 50% trifluoroacetic acid in DCM 1 × 17 min 3. DCM wash 5 × 1 min 4. 5% diisopropylethylamine in DCM 1 × 1 min 5. 5% diisopropylethylamine in NMP 1 × 1 min 6. NMP washing step 5 × 1 min 7. Addition of pre-activated protected amino acid @ (Activation by 1 equivalent of DCC and 1 equivalent of HOBt in NMP / DCM); Peptide Coupling (Part 1) 1 × 30 min 8. Addition of DMSO to the reaction mixture up to a volume fraction of 20% DMSO @ 9. Peptide Coupling (Part 2) 1 × 16 min 10. Add 3.8 equivalents of diisopropylethylamine to the reaction mixture @ 11. Peptide coupling (3rd part) 1 × 7 min 12. DCM wash 3 × 1 min 13. if the sales are incomplete repeat the clutch (back to 5.) @ 14. 10% acetic anhydride, 5% diisopropylethylamine in DCM 1 × 2 min 15. 10% acetic anhydride in DCM 1 × 4 min 16. DCM wash 4 × 1 min 17. back to 1.

b) Synthesis cycle for the Fmoc protective group technique 1. DMF washing step | 1 × 1 min 2. 20% piperidine in DMF 1 × 4 min 3. 20% piperidine in DMF 1 × 16 min 4. DMF washing step 5 × 1 min 5. Addition of pre-activated protected amino acid @ (Activation by 1 equivalent of TBTU and 1.5 equivalents of DIPEA in DMF); peptide coupling 1 × 61 min 6. DMF washing step 3 × 1 min 7. if the sales are incomplete repeat the clutch (back to 5.) @ 8. 10% acetic anhydride in DMF 1 × 8 min 9. DMF wash 3 × 1 min 10. back to 2.

II. Working up of the peptide resins obtained according to Ia

The peptide resin obtained according to Ia was dried in vacuo and into a Reaction vessel of a Teflon-HF apparatus (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 indolic formyl group, preferably ethanedithiol (0.5 ml / g Resin) was condensed under cooling with liquid N₂ hydrogen fluoride (10 ml / g resin). The mixture was allowed to warm to 0 ° and Stirred 45 minutes at this temperature. Subsequently, the hydrogen fluoride stripped off in vacuo and the residue washed with ethyl acetate, to remove remaining scavenger. The peptide was with Extracted 30% acetic acid, filtered and the filtrate lyophilized.

For the preparation of Peptidhydraziden was the peptide resin (Pam or Merrifield resin suspended in DMF (15 ml / g resin) and after addition with hydrazine hydrate (20 equivalents) for 2 days at room temperature. For workup, the resin was filtered off and the filtrate to Dry evaporated. The residue was from DMF / Et₂O or MeOH / Et₂O crystallized.

III. Working up 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 cleavage procedures (Wade, Tregear, Howard Florey Fmoc Workshop Manual, Melbourne 1985).  

The suspension of the peptide resin in the appropriate TFA mixture became Stirred at room temperature for the specified time, then the Resin filtered and washed with TFA and DCM. The filtrate and the Washing solutions were largely concentrated and the peptide by addition precipitated from diethyl ether. After cooling in an ice bath was the Precipitate filtered off, taken up in 30% acetic acid and lyophilized.  

IV. Purification and Characterization of the Peptides

Purification was carried out by 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 final products obtained was determined by analytical HPLC (Stationary phase: 100 × 2.1 mm VYDAC C-18, 5 μ, 300 Å, mobile phase logo CNRS logo INIST CH₃CN / H₂O gradient, buffered with 0.1% TFA, 40 ° C) determined. to Characterization, amino acid analysis and fast-atom bombardment Mass spectroscopy used.

B. Special working instructions Example 1 H-Leu-Ala-Asn-Gly-Val-Glu-Leu-Arg-Aso-Asn-Gln-Leu-NH₂

1.28 g of Fmoc-Leu-RINK resin (substitution ~ 0.39 mmol / g), corresponding to one Batch size of 0.5 mmol, were according to AIb with 1 mmol

Fmoc-Gln (Trt) -OH Fmoc-Gln (OtBu) -OH Fmoc-Asn (Trt) -OH Fmoc-Val-OH Fmoc-Asp (OtBu) -OH Fmoc-Gly-OH Fmoc-Arg (Pmc) -OH Fmoc-Asn (Trt) -OH Fmoc-Leu-OH Fmoc-Ala-OH Fmoc-Leu-OH

implemented.

After completion of the synthesis, the peptide-resin was deprotected N-terminal (Execution of steps 2-4 according to AIb) and then in a vacuum dried; the yield was 2.2 g.

1.1 g of the thus obtained resin was subjected to TFA cleavage according to AIII. The crude product (270 mg) was purified by gel filtration (SEPHADEX® G-10) and medium pressure chromatography (see AIV, 40-60% A, 0.25% min ^-1 ).

example 2 H-Arg-Arg-Ala-Asn-Ala-Leu-Leu-Ala-Asn-Gly-Val-Glu-Leu-Arg-Asp-Asn-Gly-Leu- Val-Val-Pro-Ser-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 prepared according to AIb with 1 mmol each

implemented.

After completion of the synthesis of the N-terminus was deprotected (execution of the Steps 2-4 according to AIb). The obtained peptide-resin was vacuum dried; the yield was 1.2 g.

The crude peptide (584 mg) obtained after TFA cleavage according to AIII was purified by gel filtration (SEPHADEX® G-10) and medium pressure chromatography (see AIV, 40-60%, 0.25% min ^-1 ). There were obtained 180 mg of pure product.

Analogously to Example 1 and 2 can be prepared: (to the coupling of N-methyl-amino acids were used as coupling reagent by PyBroP):

3. H-Leu-Ala-Asn-Gly-Val-Glu-Leu-Arg-Asp-Asn-Gln-Leu-OH
4. H-Leu-Ala-Asn-Gly-MeVal-Glu-Leu-Arg-Asp-Asn-Gln-Leu-OH
5. H-Leu-Ala-Asn-Gly-Val-Glu-MeLeu-Arg-Asp-Asn-Gln-Leu-OH
6. H-Ala-Asn-Gly-Val-Glu-Leu-Arg-Asp-Asn-Gln-OH
7. H-Leu-Ala-Asn-Sar-Val-Glu-Leu-Arg-Asp-Asn-Gln-Leu-OH
8. H-Ala-Leu-Leu-Ala-Asn-Gly-Val-Glu-Leu-Arg-Asp-Asn-Gly-Leu-Val-OH
9. Ac-Arg-Arg-Ala-Asn-Ala-Leu-Leu-Ala-Asn-Gly-Val-Glu-Leu-Arg-Asp-Asn-Gly-Leu-Val-Val-Pro-Ser-Glu OH

example 10

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

implemented.

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

The obtained peptide resin was dried in vacuo; the yield was 1.79 g.

0.9 g of the resulting resin was subjected to HF cleavage according to AII subjected. The lyophilized crude product became 1% in 2 liters Acetic acid was added and the pH then with aqueous ammonia 8.4 set. Under argon atmosphere was slowly 0.01 n K₃ [Fe (CN) ₆] solution is added dropwise until the yellowish-green coloration longer than 15 min persisted. It was stirred for a further 1 h, then with glacial acetic acid acidified to pH 4.5 and with 15 ml of an aqueous suspension of a Anionenaustauschers (BIORAD® 3 × 4A, chloride form). 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 give prevent possible oxidation of the free cysteine residues.

The crude product was purified by gel chromatography (SEPHADEX® G-15). There were obtained 78 mg of pure product.

Analogously to Example 10 can be prepared (for the synthesis of corresponding peptide acids, Boc-Cys (pMB) -PAM resin was used):  

example 30

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

implemented. After completion of the synthesis, the N-terminus was acetylated (Execution of steps 2-4 and 8-9 according to AIb). The peptide-resin became dried in vacuum; the yield was 1.95 g.

The crude product (720 mg) obtained after TFA cleavage according to AIII was dissolved in 500 ml of degassed DMF. After addition of 250 mg NaHCO₃ and 440 mg BOP was stirred for 3 days at room temperature. It was then evaporated to dryness and the crude peptide purified by gel chromatography (SEPHADEX LH 20). The isolated monomer (370 mg) was subjected to HF cleavage according to AII and the deprotected peptide was purified by medium chromatography (see AIV, 55 to 75% A, 0.25% min ^-1 ). There were obtained 83 mg of pure product

Analogously to Example 30 can be produced:

example 40

1.09 g of Fmoc-Gly-p-alkoxybenzyl alcohol resin (substitution ~ 0.46 mmol / g), according to a batch size of 0.5 mmol, according to AIb with 2 mmol

Fmoc-Asn (Trt) -OH Fmoc-Asp (OChx) -OH Fmoc-Ala-OH Fmoc-Arg (Tos) -OH Fmoc-Leu-OH Fmoc-Glu (OBzl) -OH Fmoc-Val-OH

implemented. Upon completion of the synthesis, the peptide-resin became N-terminal deprotected (execution of steps 2-4 according to AIb) and then in Vacuum dried. The yield was 1.43 g.

The crude peptide obtained after TFA cleavage according to AIII was dissolved in 500 ml of degassed DMF. After addition of 210 mg NaHCO₃ and 440 mg BOP was stirred for three days at room temperature. It was then evaporated to dryness and the crude peptide was purified by gel chromatography (SEPHADEX® LH 20). The isolated monomer (150 mg) was subjected to HF cleavage according to AII and the deprotected peptide was purified by medium pressure chromatography (see AIV, 55 to 75% min ^-1 ). There were obtained 37 mg of pure product.

Analogously to Example 40 can be produced:

example 44

1.2 g of resin according to Rink et al. (NOVABIOCHEMI) according to a batch size of 0.6 mmol was according to AIb with 2 mmol  

implemented. After completion of the synthesis, the N-terminus was acetylated (Execution of steps 2 to 4 and 8 to 9 according to AIb). The peptide resin was dried in vacuo; Yield 3.05 g.

The crude peptide (975 mg) obtained after TFA cleavage according to AIII was analyzed in 3 l of 0.1% acetic acid and the pH then with aqueous Ammonia adjusted to 8.4. Under argon atmosphere was slowly 0.01 n K₃ [Fe (CN) ₆] solution was added dropwise. It was stirred for 1 h, then with Acetic acid acidified to pH 4.5 and with 15 ml of an aqueous suspension an anion exchanger (BIORAD 3 × 4A, chloride form). To The ion exchange resin was filtered off for 30 minutes and the filtrate was filtered off with suction HD-Sil C-19 RP column, washed several times with water and the Product finally eluted with methanol and by gel chromatography (SEPHADEX® G25).

The cyclized product (420 mg) was subjected to HF cleavage according to AII. The lyophilized crude product was cycled as described above and purified by gel chromatography (SEPHADEX® G-25) and medium pressure chromatography (see AIV, 55 to 75% A, 0.25% min ^-1 ). There were obtained 38 mg of pure product.

Analogously to Example 44 can be prepared:

example 46

1.2 g of resin according to Rink et al. (NOVABIOCHEMI) according to one Batch size of 0.6 mmol was according to AIb with 2 mmol

implemented. After completion of the synthesis, the N-terminus was acetylated (Execution of steps 2 to 4 and 8 to 9 according to AIb). The peptide resin was dried in vacuo; Yield 2.96 g.

The crude peptide (853 mg) obtained after TFA cleavage according to AIII was dissolved in 500 ml of degassed DMF. After addition of 240 mg NaHCO₃ and 440 mg BOP was stirred for 3 days at room temperature. It was then evaporated to dryness and the crude peptide was purified by gel chromatography (SEPHADEX® LH 20). The isolated monomer (395 mg) was subjected to HF cleavage according to AII, taken up in 3 L of 1% acetic acid and the pH adjusted to 8.4 with aqueous ammonia. Under argon atmosphere slowly 0.01 N K₃ [Fe (CN) ₆] solution was added dropwise. The mixture was stirred for a further 1 h, then acidified to pH 4.5 with glacial acetic acid and treated with 15 ml of an aqueous suspension of an anion exchanger (BIORAD 3 × 4A, chloride form). After 30 minutes, the ion exchange resin was filtered off, the filtrate was placed on a HD-Sil C-19 RP column, washed several times with water and the product finally eluted with methanol. The bicyclic crude product was purified by gel chromatography (SEPHADEX® G25) and medium pressure chromatography (see AIV, 55 to 75% A, 0.25% min ^-1 ). There were obtained 86 mg of pure product.

Analogous to Example 46 can be produced

example 50 Ac-Leu-Ala-Asn-Gly-Val-Glu-NH-CH [CH₂CH (CH₃) ₂] -Arg-Asp-Asn-Gln-Leu-NH-₂

0.8 g of resin according to Rink et al. (NOVABIOCHEM) according to one Batch size of 0.4 mmol was according to AIb with 1 mmol

Fmoc-Leu-OH Fmoc-Asp (OtBu) -OH Fmoc-Gln (Trt) -OH Fmoc-Arg (Pmc) -OH Fmoc-Asn (Trt) -OH

implemented. After cleavage of the Fmoc protecting group (execution of the Steps 2 to 4 according to AIb), the resin in DMF, the 1% acetic acid suspended, 3 equivalents of Fmoc-Leucinal (prepared from Fmoc-leucine O, N-dimethylhydroxamate according to Castro, B., Fehrentz, J.-A. (1983) Synthesis, 676-678) and 3 equivalents of NaBH₃CN. After 3 h (negative ninhydrin test) was washed intensively and according to AIb with each 1 mmol

Fmoc-Glu (OtBu) -OH Fmoc-Val-OH Fmoc-Asn (Trt) Fmoc-Gly-OH Fmoc-Ala-OH Fmoc-Leu-OH

implemented. After completion of the synthesis, the N-terminus was acetylated (Execution of steps 2 to 4 and 8 to 9 according to AIb). The peptide resin was dried in vacuo (yield: 1.55 g).

The thus-obtained resin was subjected to TFA cleavage according to AIII. The crude product was purified by gel filtration (SEPHADEX® G-25) and medium pressure chromatography (see AIV, 40 to 60% A, 0.25% min ^-1 ) (yield 105 mg).

Analogously to Example 50 can be produced:

51. Ac-Leu-Ala-Asn-NH-CH₂-CH₂-Val-Glu-Leu-Arg-Asp-Asn-Gln-Leu-NH₂
52. Ac-Leu-Ala-Asn-Gly-NH-CH [CH (CH₃) ₂] -CH₂-Glu-Leu-Arg-Asp-Asn-Gln-Leu-N-H₂
53. Ac-Arg-Arg-Ala-Asn-Ala-Leu-Leu-Ala-Asn-Gly-NH-CH [CH (CH₃) ₂] -CH₂-Glu-Leu-Arg-Asp-Asn-Gly-Leu -Val-Val-Pro-Ser-Glu-NH₂

Claims (10)

1. Compounds of the formula I wherein A, B, D, E and J are hydrogen atoms or one of the groups (with b meaning a number from 1 to 6 and T meaning OH, CH₃S-, (CH₃) ₂CH-, C₆H₅-, p-HO-C₆H₄-, HS-, HO-CO-, H₂N- CO- or H₂N-C (= NH) -NH group)
F Asp, Asn or His means
T¹-T⁶ are hydrogen atoms or methyl groups,
Z¹-Z⁶ each represent an oxygen atom or two hydrogen atoms
X represents the groups G-, G-NH-CHM-CO-, G-NH-CHM-CO-W-, GR-NH-CHM-CO- or GR-NH-CHM-CO-W- and
Y -P, -NH-CHQ-CO-P, -V-NH-CHQ-CO-P, -NH-CHQ-CO-UP, -V-NH-CHQ-CO-UP,
-NH-CHQ-K-NH-CHL-CO-P, -V-NH-CHQ-K-NH-CHL-CO-P,
Is -NH-CHQ-K-NH-CHL-CO-UP or -V-NH-CHQ-K-NH-CHL-CO-UP,
where in X and Y
G represents a hydrogen atom or an amino-protecting group,
P is an OH or NH₂ group or a carboxyl protecting group or
G and P together also represent a covalent bond or the group -CO- (CH₂) _a -NH-, where a is a number from 1 to 12,
K, R, U, V and W are peptide chains of 1-5 naturally occurring α-amino acids,
L, M and Q are hydrogen atoms or one of the groups
-CH₃, -CH₂-CH (CH₃) ₂, -CH₂-CO-NH₂, - (CH₂) ₃-NH-C (NH) -NH₂, - (CH₂) ₂-CO-NH₂,
-CH (CH₃) ₂, -CH₂OH, - (CH₂) ₂COOH, or
A, B, D, M, L and Q in pairs one or two
- (CH₂) _c -SS- (CH₂) _d -
- (CH₂) _e -CO-NH- (CH₂) _f - or - (CH₂) _e -NH-CO- (CH₂) _g -NH-CO- (CH₂) _f -Bridge (s)
(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 P is a hydroxy or amino group or a carboxyl-protecting group represent and A, B, D, M, L and Q not with each other are connected. 3. Peptides according to claim 1, wherein G represents a hydrogen atom or an amino protecting group and P represents a hydroxy or amino group or a carboxyl-protecting group, and M and Q, A and Q, B and Q or D and Q together form a - (CH₂) _c -SS - (CH₂) _d bridge mean. 4. Peptides according to claim 1, in which G represents a hydrogen atom or an amino-protecting group and P represents a hydroxyl or amino group or a carboxyl-protecting group and M and Q, A and Q, B and Q or D and Q together form a group - (CH₂) _e - NH-CO- (CH₂) _f or - (CH₂) _e -NH-CO- (CH₂) _g -NH-CO- (CH₂) _f . 5. Peptides according to claim 1, wherein G + P together represent a covalent bond or -CO- (CH₂) _a -NH-. 6. Peptides according to claim 1, wherein G represents a hydrogen atom or an amino-protecting group and P represents a hydroxy or amino group or a carboxyl-protecting group and either M + D and A + Q or A + L and D + Q together
- (CH₂) _c -SS- (CH₂) _d - or - (CH₂) _e -NH-CO- (CH₂) _f bridges (with e and f in the meaning of a number from 1 to 4). 7. Peptides according to claim 1, wherein G is a hydrogen atom or a Amino protecting group and P is a hydroxy or amino group or a Represent carboxyl protecting group and Z¹ to Z⁶ each represent two hydrogen atoms mean. 8. peptides according to claim 1 to 7 for use in the control of Diseases. 9. Use of the peptides according to claims 1 to 7 for controlling neoplastic diseases and autoimmune diseases as well as Combat and prophylaxis of infections, inflammation and Rejection reactions in transplants. 10. A process for the preparation of the peptides according to claim 1 to 7, characterized characterized in that they are known in peptide chemistry Produces methods.