WO1992011285A1

WO1992011285A1 - Novel tnf peptides

Info

Publication number: WO1992011285A1
Application number: PCT/EP1991/002405
Authority: WO
Inventors: Hans-Joachim Boehm; Lothar Daum; Andreas Haupt; Dagmar Pettig; Nigel Walker; Daniela Maennel; Reiner Frank; Rainer Stiemer
Original assignee: Basf Aktiengesellschaft
Priority date: 1990-12-21
Filing date: 1991-12-13
Publication date: 1992-07-09
Also published as: DE4041187A1

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

New TNF peptides

The invention relates to new peptides derived from tumor necrosis factor (TNF), their production and their use as medicaments.

By Carswell et al. (Proc. Natl. Acad. Sci. USA 72, 3666, 1975), it has been reported that the serum of endotoxin-treated animals previously infected with the Mycobacterien-Stairrm Calmette-Guerin (BCG) caused hemorrhagic necrosis in various Caused tumors in the mouse. This activity was attributed to the tumor necrosis factor. TNF also shows cytostatic or cytotoxic activity against a variety of transformed cell lines in vitro, whereas normal human and animal cell lines are not affected

(Lymphokine Reports Vol. 2, pp 235-275, Academic Press, New York, 1981). Biochemical characterization and the gene for human TNF have recently been described (Nature 312, 724, 1984;

J. Biol. Chem. 260, 2345, 1985; Nucl. Acids Res. 13, 6361, 1985).

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

ValArgSerSerSerArgThrProSerAspLysProValAlaHisValValAlaAsnPro

GlnAlaGluGlyGlnLeuGlnTrpLeuAsnArgArgAlaAsnAlaLeuLeuAlaAsnGly ValGluLeuArgAspAsnGlnLeuValValProSerGluGlyLeuTyrLeuIleTyrSer

GlnValLeuPheLysGlyGlnGlyCysProSerThrHisValLeuLeuThrHisThrlle

SerArglleAlaValSerTyrGlnThrLysValAsnLeuLeuSerAlalleLysSerPro

CysGlnArgGluThrProGluGlyAlaGluAlaLysProTrpTyrGluProlleTyrLeu

GlyGlyValPheGlnLeuGluLysGlyAspArgLeuSerAlaGluIleAsnArgProAsp TyrLeuAspPheAlaGluSerGlyGlnValTyrPheGlyllelleAlaLeu Furthermore, the TNF gene of 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 in inflammatory reactions (Pharmac. Res. 5, 129, 1988). The involvement of TNF in septic shock (Science 229, 869, 1985) and graft versus host disease (J. Exp. Med. 166, 1280, 1987) was shown in the animal model.

It has now been found that peptides with a significantly lower molecular weight have favorable properties.

The invention relates to compounds of the formula I.

wherein

A, B, D, E and 3 hydrogen atoms or one of the groups

-CH (CH ₃ ) ₂ , -CH (CH ₃ ) -C ₂ H ₅ , -C ₆ H ₅ , -CH (OH) -CH ₃ ,

or - (CH ₂ ) _b -T

(with b meaning a number from 1 to 6 and T meaning OH-, CH ₃ S-, (CH ₃ ) ₂ CH-,

C ₆ H ₅ -, p-HO-C ₆ H ₄ -, H ₂ N, HS, HO-CO, H ₂ N-CO or H ₂ NC (= NH) -NH group)

F means Asp, Asn or His,

T ¹ -T ^{6 are} hydrogen atoms or methyl groups,

Z ¹ -Z ⁶ each represent an oxygen atom or two hydrogen atoms

X the groups G-, G-NH-CHM-CO-, G-NH-CHM-CO-W-,

GR-NH-CHM-CO- or GR-NH-CHM-CO-W- means and Y -P, -NH-CHQ-CO-P, -V-NH-CHQ-CO-P,

-NH-CHQ-CO-U-P, -V-NH-CHQ-CO-U-P, -NH-CHQ-K-NH-CHL-CO-P,

-V-NH-CHQ-K-NH-CHL-CO-P,

-NH-CHQ-K-NH-CHL-CO-U-P or

-V-NH-CHQ-K-NH-CHL-CO-U-P, where in X and Y G is a hydrogen atom or a

Amino protecting group means

P for an OH or NH ₂ group or

Carboxyl protecting group stands or

G and P together also mean a covalent bond or the group -CO- (CH ₂ ) _a -NH-, where a is a number from 1 to 12, K, R, U, V and W peptide chains from 1-5 naturally occurring represent α-amino acids,

L, M and Q are hydrogen atoms or one of the groups -CH ₃ , -CH ₂ -CH (CH ₃ ) ₂ , -CH ₂ -CO-NH ₂ ,

- (CH ₂ ) ₃ -NH-C (NH) -NH ₂ , - (CH ₂ ) ₂ -CO-NH ₂ ,

-CH (CH ₃ ) ₂ , -CH ₂ OH, - (CH ₂ ) ₂ COOH, are or

A, B, D, M, L and Q in pairs one or two

- (CH ₂ ) _c -SS- (CH ₂ ) _d -,

- (CH ₂ ) _e -CO-NH- (CH ₂ ) _f - or

- (CH ₂ ) _e -NH-CO- (CH ₂ ) g-NH-CO- (CH ₂ ) _f bridge (s) (with c and d meaning a number from 1 to 4, e and f one Number from 1 to 6 and g of a number from 1 to 12), and their salts with physiologically tolerated acids.

The peptides of formula I are made up of L-amino acids, but they can contain 1 to 2 D-amino acids or N-methyl-amino acids. The side chains of the trifunctional amino acids can carry protective groups or be unprotected. In particular, the following can be mentioned as physiologically compatible 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; P = OH, NH ₂ , carboxyl protecting group, A, B, D, M, L, Q not connected to each other), disulfide bridged (G = H, amino protecting group; P = OH, NH ₂ , carboxyl protecting group; A + Q, B + Q, D + Q or M + Q = - (CH ₂ ) _c -SS- (CH ₂ ) _d -),

Side chain bridged (G = H, amino protecting group, P = OH, NH ₂ , carboxyl protecting group, A + Q, B + Q, D + Q or

M + Q = - (CH ₂ ) _e -NH-CO- (CH ₂ ) _f - or

- (CH ₂ ) _e -NH-CO- (CH ₂ ) g-NH-CO- (CH ₂ ) _f -), head-to-tail linked (G + P = covalent bond or -CO- (CH ₂ ) _a -NH-) OH, NH ₂ ,

Carboxyl protecting group) or bicyclic (G = H, amino protecting group; P = OH, NH ₂ , carboxyl protecting group, M + D and A + Q or A + L and D + Q each correspond to - (CH ₂ ) _c -SS- (CH ₂ ) _d bridges or

- (CH ₂ ) _e -NH-CO- (CH ₂ ) _f bridges), or contain reduced peptide bonds. The new compounds can be prepared by methods known in peptide chemistry.

So you can build the peptides sequentially from amino acids or by fragment linking of suitable small peptides. In the sequential construction, the peptide chain is gradually extended by one amino acid each, starting at the C terminus. In the

Fragment coupling can link fragments of different lengths to one another, the fragments in turn by sequential construction from amino acids or in turn by

Fragment coupling can be obtained. The cyclic peptides are obtained after synthesis of the open-chain peptides by a cyclization reaction carried out in high dilution. Both in the sequential structure and in the fragment coupling, the building blocks must be linked by forming an amide bond. Enzymatic and chemical methods are suitable for this.

Chemical methods for forming amide bonds are dealt with in detail by 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. The azide method, the symmetrical and mixed anhydride method, in situ generated or preformed active esters and the amide bond formation with the aid of coupling reagents (activators), in particular dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-ethoxycarbonyl-2-ethoxy-1, are particularly preferred. 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-C1 ), Diphenylphosphoryl azide (DPPA), Castro's reagent (BOP), O-benzotriazolyl-N, N, N ', N'-tetramethyluronium salts (TBTU), 2,5-diphenyl-2,3-dihydro-3-oxo 4-hydroxythiophene dioxide (Steglich reagent; HOTDO),

Bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBroP) and 1,1'-carbonyl-diimidazole (CDI). The coupling reagents can 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 .

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 α-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 chain can be in solution, in suspension or by a similar method as described by Merrifield in 3 Amer. Chem. Soc. 85, 2149, 1963. Particular preference is given to 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

Polymers are bound, which are only insoluble in the solvents used and must have a stable physical form that enables easy filtration. The polymer must contain a functional group to which the first protected amino acid can be bound by a covalent bond. Various polymers are suitable for this purpose, e.g. 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 resin, resin according to Breipohl et al. (Tetrahedron Lett. 28, 565, 1987; Fa. BACHEM), HYCRAM resin (ORPEGEN), SASRIN resin (BACHEM) (dimethoxyphenylaminomethyl) phenoxy resin, RINK resin; (Tetrahedron Lett. 28, 3787 (1987, Novabiochem) or o-chloro-trityl resin (BlOHELLAS).

All solvents which prove inert under the reaction conditions are suitable for peptide synthesis in solution, in particular 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 on the polymeric support can be carried out in all inert organic solvents in which the amino acid derivatives used are soluble; however, preference is given to solvents which additionally have resin-swelling properties, such as DMF, DCM, NMP,

Acetonitrile and DMSO, as well as mixtures of these solvents.

After successful synthesis, the peptide is split off from the polymeric carrier. The conditions under which the various types of resin can be split off are known from the literature. Acid and palladium-catalyzed cleavage reactions are most common

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 as e.g. Morpholine. Depending on the choice of the protective groups, these can be retained under the cleavage conditions or can also be split off. Partial deprotection of the peptide can also be useful if certain derivatization reactions or a cyclization are to be carried out.

Some of the new peptides show TNF activity. These are TNF agonists. Another part of the peptides has a high affinity for the cellular TNF receptor, but without having an activity comparable to TNF. So you pose

TNF antagonists. In competition with natural TNF, they bind to the cellular TNF receptor and thus suppress the

TNF effect. The new peptides prove to be valuable Medicines used to treat neoplastic and autoimmune diseases, as well as to fight and prevent infections, inflammation and rejection

Transplants can be used.

The characterization of the new peptides on their agonistic or antagonistic effect was carried out essentially in the following test systems: I. Receptor binding test

II. Monocyte Activation Test: Induction of TNFmRNA

III. Granulocyte activation test: release of oxygen radicals

IV. Cytotoxicity test ad I. Receptor binding test

Both the agonistic and the antagonistic effects of peptides require that the latter bind to the TNF receptor. This means that peptides with agonistic or antagonistic activity and rHu-TNF bind to the TNF receptor

TNF-sensitive indicator cells compete. On

Competition receptor binding test (int. J. Cancer 38, 127 (1986) was carried out as follows:

Using human U937 cells (2 × 10 ⁶ cells per group, 300 μl assay volume) and 1 ng ¹²⁵ I-labeled rHuTNF (lactoperoxidase method according to Bolton), the

Determined peptide concentration, which leads to a 50% displacement of the presented ¹²⁵ I-rHuTNF from the TNF receptor. ad II. Monocyte activation test

The agonistic evaluation of the new peptides is based on their TNFmRNA inducing effect in human monocytes. The test (J. Immunol. 144, 1144 (1990) was carried out as follows:

Human peripheral blood monocytes (5 to 10 × 10 ⁶ per group, 3 ml assay volume) were incubated with the peptides at 37 ° C. for 2 hours. RNA was extracted and the induction of TNF mRNA was measured by Northern blot analysis. The antagonistic evaluation of the new peptides is based on their ability to inhibit rHuTNF-induced induction of TNFmRNA in this test system. ad III: granulocyte activation test

The agonistic effect of the new peptides is based on their induction of radical oxygen release (oxygen burst) in human granulocytes. The test (Methods in Enzymology 133, 449 (1986) was carried out as follows:

Using polymorphonuclear neutrophils (2.5 × 10 ⁵ per group, 500 μl assay volume), the peptide-induced release of radical oxygen was measured in a chemiluminescence test.

The antagonistic evaluation of the new peptides is based on their ability to inhibit the rHuTNF-induced radical oxygen release in this test system. ad IV: cytotoxicity test

The agonistic evaluation of the new peptides is based on their cytotoxic effects on TNF-sensitive tumor cells. The test was carried out as follows:

Using human MCF-7 cells (2 × 10 ⁴ cells per group, 200 μl assay volume), the peptide concentration was determined in a proliferation test. After 24 hours of incubation, the incorporation of ³ H-τdR into the DNA of the MCF-7 cells was determined 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 abbreviated in the examples with the well-known three-letter code. In addition,

Ac = acetic acid, Sar = sarcosine. A. General work regulations

I. The synthesis of the peptides according to claim 1 was carried out using the standard methods of solid-phase peptide synthesis on a fully automatic peptide synthesizer model 430A from APPLIED BIOSYSTEMS. The device uses different synthesis cycles for the Boc and Fmoc protection group technology. a) Synthesis cycle for Boc protection group technology

1. 30% trifluoroacetic acid in DCM 1 × 3 min

2. 50% trifluoroacetic acid in DCM 1 x 17 min

3. DCM wash 5 × 1 min

4. 5% diisopropylethylamine in DCM 1 x 1 min. 5% diisopropylethylamine in NMP 1 x 1 min

6. NMP wash 5 × 1 min

7. Add the pre-activated protected

Amino acid (activation by 1 equivalent of DCC and 1 equivalent of HOBt in

in NMP / DCM); Peptide coupling (1st part) 1 × 30 min

8. Add DMSO to the reaction mixture

up to a volume share of 20% DMSO

9. Peptide coupling (2nd part) 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. Repeat for incomplete sales

the clutch (back to 5.)

14. 10% acetic anhydride, 5% diisopropylethylamine in DCM 1 x 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 Fmoc protection group technology

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 wash 5 × 1 min 5. Add the pre-activated protected

Amine acid (activation by 1 equivalent of TBTU and 1.5 equivalent of DIPEA

in DMF); Peptide coupling 1 × 61 min 6. DMF washing step 3 × 1 min

7. Repeat for incomplete sales

the clutch (back to 5.)

8. 10% acetic anhydride in DMF 1 x 8 min

9. DMF washing step 3 × 1 min 10. back to 2. Working up 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

(PENINSULA) transferred. After adding a scavenger, preferably anisole (1 ml / g resin) and, in the case of tryptophan-containing peptides, a thiol to remove the indole formyl group, preferably ethanedithiol

(0.5 ml / g resin) was cooled with liquid N ₂

Hydrogen fluoride condensed (10 ml / g resin). The mixture was allowed to warm to 0 ° C and stirred at this temperature for 45 min. The hydrogen fluoride was then stripped off in vacuo and the residue was washed with ethyl acetate in order to remove remaining scavengers. The peptide was extracted with 30% acetic acid, filtered and the filtrate lyophilized.

To produce peptide hydrazides, the peptide resin (Pam or Merrifield resin) was suspended in DMF (15 ml / g resin) and, after addition with hydrazine hydrate (20 equivalents), stirred for 2 days at room temperature. For working up, the resin was filtered off and the filtrate was evaporated to dryness. The residue was crystallized from DMF / Et ₂ O or MeOH / Et ₂ O. 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, subjected to one of the following cleavage 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 indicated time, then the resin was filtered off and washed with TFA and DCM. The filtrate and the washing solutions were largely concentrated and the peptide was precipitated by adding diethyl ether. After cooling in an ice bath, the precipitate was filtered off, 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 / H ₂ O).

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 = CH ₃ CN / H ₂ O gradient, buffered with 0.1% TFA, 40 ° C.). to

Amino acid analysis and characterization

Fast atom bombardment mass spectroscopy. B. Special working regulations

example 1

H-Leu-Ala-Asn-Gly-Val-Glu-Leu-Arg-Asp-Asn-Gln-Leu-NH ₂

1.28 g Fmoc-Leu-RINK resin (substitution - 0.39 mmol / g), corresponding to a batch size of 0.5 mmol, were mixed according to Alb 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 the synthesis was complete, the peptide-resin was deprotected at the N-terminal (steps 2-4 according to Alb) and then dried in vacuo; the yield was 2.2 g. 1.1 g of the resin thus obtained were subjected to TFA cleavage in accordance with AIII. The crude product (270 mg) was purified by gel filtration (SEPHADEX® G-10) and medium pressure chromatography (cf. 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-AspAsn-Gly-Leu-Val-Val-Pro-Ser-Glu-OH 0.46 g Fmoc-Glu (OtBu) -p-Alkoxybenzylalkoholharz (substitution ~ 0.55 mmol / g), corresponding to a batch size of 0.25 mmol, were according to Alb with 1 mmol

Fmoc-Ser (OtBu) -OH Fmoc-Asn (Trt) -OH Fmoc-Asn (Trt) -OH

Fmoc-Pro-OH Fmoc-Asp (OtBu) -OH Fmoc-Ala-OH

Fmoc-Val-OH Fmoc-Arg (PMc) -OH Fmoc-Leu-OH

Fmoc-Val-OH Fmoc-Leu-OH Fmoc-Leu-OH

Fmoc-Leu-OH Fmoc-Glu (OtBu) OH Fmoc-Ala-OH

Fmoc-Gly-OH Fmoc-Val-OH Fmoc-Asn (Trt) -OH

Fmoc-Gly-OH Fmoc-Ala-OH

Fmoc-Arg (PMc) -OH

Fmoc-Arg (PMc) -OH implemented.

After the synthesis, the N-terminus was deprotected

(Execution of steps 2-4 according to Alb). The received

Peptide resin was dried in vacuo; the yield was 1.2 g. The crude peptide (584 mg) obtained after the 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 ) cleaned. 180 mg of pure product were obtained. Analogously to Examples 1 and 2, the following can be prepared: (PyBroP was used as a coupling reagent for coupling the N-methyl amino acids

used):

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-LeuVal-OH

9. Ac-Arg-Arg-Ala-Asn-Ala-Leu-Leu-Ala-Asn-Gly-Val-Glu-Leu-ArgAsp-As n-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 in accordance with Ala with 2 mmol of Boc-Val-OH Boc-Arg (Tos ) -OH Boc-Asn-OH

Boc-Val-OH Boc-Leu-Oh Boc-Ala-OH

Boc-Leu-OH Boc-Glu (OBzl) -OH

Boc-Gln-OH Boc-Val-OH

Boc-Asn-OH Boc-Cys (pMB) -OH

Boc-Asp (OCHx) -OH implemented.

After the synthesis was complete, the N-terminus was acetylated

(Execution of steps 1 - 6 and 14 - 16 according to Ala).

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

0.9 g of the resin thus obtained was subjected to HF cleavage in accordance with AII. The lyophilized crude product was taken up in 2 1 0.1% acetic acid and the pH was then with aqueous

Ammonia set to 8.4. 0.01 N K ₃ [Fe (CN) ₆ ] solution was slowly added dropwise under an argon atmosphere until the yellowish green Staining persisted for more than 15 min. Stirring was continued for 1 h, then acidified with glacial acetic acid to pH 4.5 with 15 ml of an aqueous suspension of an anion exchanger (BIORAD ^® 3 x 4A, chloride form) was added. After 30 minutes, the ion exchange resin was filtered off, the filtrate was concentrated to 100 ml on a rotary evaporator 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). 78 mg of pure product were obtained. The following can be prepared analogously to Example 10 (Boc-Cys (pMB) -PAM resin was used for the synthesis of the corresponding peptide acids):

Example 30

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

Fmoc-Lys (Boc) -OH Fmoc-Gln- (Trt) -OH Fmoc-Leu-OH

Fmoc-Val-OH Fmoc-Asn (Trt) -OH Fmoc-Glu (OBzl) -OH Fmoc-Val-OH Fmoc-Asp (OChx) -OH Fmoc-Val-OH

Fmoc-Leu-OH Fmoc-Arg (Tos) -OH Fmoc-Glu (OtBu) -OH

Fmoc-Asn (Trt) -OH

Fmoc-Ala-OH implemented. When synthesis was complete, the N-terminus

acetylated (steps 2 - 4 and 8 - 9 according to Alb). The peptide resin was dried in vacuo; the yield was 1.95 g.

The crude product obtained after the TFA cleavage according to AIII

(720 mg) was dissolved in 500 ml degassed DMF. After adding 250 mg of NaHCO ₃ and 440 mg of BOP, the mixture was stirred at room temperature for 3 days. The mixture was then evaporated to dryness and the crude peptide was purified by gel chromatography (SEPHADEX LH 20). The isolated monomers (370 mg) were subjected to an HF cleavage according to All and the deprotected peptide was purified by medium pressure chromatography (cf. AIV; 55 to 75% A; 0.25% min ^-1 ). 83 mg of pure product were obtained.

Example 40

1.09 g Fmoc-Gly-p-alkoxybenzyl alcohol resin

(Substitution ~ 0.46 mmol / g), corresponding to a batch size of 0.5 mmol, was according to Alb with 2 mmol

Fmoc-Asn- (Trt) -OH Fmoc-Asp (QChx) -OH

Fmoc-Ala-OH Fmoc-Arg (Tos) -OH

Fmoc-Leu-OH

Fmoc-Glu (0Bzl) -0H

Fmoc-Val-OH implemented. After synthesis was complete, the peptide resin

Deprotected at the N terminal (steps 2 - 4 according to Alb) and then dried in vacuo. 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 adding 210 mg of NaHCO ₃ and 440 mg of BOP, the mixture was stirred at room temperature for three days. The mixture 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 an HF cleavage according to All and the deprotected peptide was purified by medium pressure chromatography (cf. AIV; 55 to 75% A; 0.25% min ^-1 ). 37 mg of pure product were obtained.

Analogously to example 40, the following can be produced:

η

Example 44

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

Fmoc-Gly-OH Fmoc-Gln (Trt) -θH Fmoc-Val-OH

Fmoc-Glu (OtBu) -OH Fmoc-Asn (Trt) -OH Fmoc-Cys (pMB) -0H

Fmoc-Ser (tBu) -OH Fmoc-Asp (OtBu) -OH Fmoc-Asn (Trt) -OH

Fmoc-Cys (pMB) -OH Fmoc-Arg (Pmc) -OH Fmoc-Ala-OH

Fmoc-Val-OH Fmoc-Leu-OH Fmoc-Cys (Trt) -OH

Fmoc-Val-OH Fmoc-Cys (Trt) -OH Fmoc-Leu-OH

Fmoc-Leu-OH implemented. When synthesis was complete, the N-terminus

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 the TFA cleavage according to AIII was taken up in 3 liters of 0.1% acetic acid and the pH was then adjusted to 8.4 with aqueous ammonia. 0.01 N K ₃ [Fe (CN) ₆ ] solution was slowly added dropwise under an argon atmosphere. The mixture was stirred for a further 1 h, then acidified to pH 4.5 with glacial acetic acid and 15 ml of an aqueous suspension of an anion exchanger (BIORAD 3 × 4A, chloride form) were added. After 30 min the ion exchange resin was filtered off, the filtrate was placed on an HD-Sil C-19 RP column, washed several times with water and the product was finally eluted with methanol and purified by means of gel chromatography (SEPHADEX ^® G25).

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

Fmoc-Gly-OH Fmoc-Gln (Trt) -OH Fmoc-Val-OH

Fmoc-Glu (OBzl) -OH Fmoc-Asn (Trt) -OH

Fmoc-Cys (pMB) -OH

Fmoc-Ser (Bzl) -OH Fmoc-Asp (OChx) -OH Fmoc-Asn (Trt) -OH

Fmoc-Cys (pMB) -OH Fmoc-Arg (Tos) -OH Fmoc-Ala-OH

Fmoc-Val-OH Fmoc-Leu-OH Fmoc-Asp (OtBu) -OH

Fmoc-Val-OH Fmoc-Lys (Boc) -OH Fmoc-Leu-OH

Fmoc-Leu-OH implemented. When synthesis was complete, the N-terminus

acetylated (execution of steps 2 to 4 and 8 to 9 according to Alb). The peptide resin was dried in vacuo; Yield 2.96 g.

The crude peptide (853 mg) obtained after the TFA cleavage according to AIII was dissolved in 500 ml degassed DMF. After adding 240 mg of NaHCO3 and 440 mg of BOP, the mixture was stirred at room temperature for 3 days.

The mixture was then evaporated to dryness and the crude peptide passed through

Gel chromatography (SEPHADEX ^® LH 20) cleaned. The isolated monomer (395 mg) was subjected to HF cleavage according to All, taken up in 3 1 1% acetic acid and the pH was adjusted to 8.4 with aqueous ammonia. 0.01 N K ₃ [Fe (CN) ₆ ] solution was slowly added dropwise under an argon atmosphere. It was still 1 h

stirred, then acidified to pH 4.5 with glacial acetic acid and mixed with 15 ml of an aqueous suspension of an anion exchanger (BIORAD 3 × 4A, chloride form). After 30 min

Filtered ion exchange resin, the filtrate was placed on an HD-Sil C-19 RP column, washed several times with water and the product was finally eluted with methanol. The bicyclic

Crude product was purified by means of gel chromatography (SEPHADEX ^® 625) and medium pressure chromatography (cf. AIV; 55 to 75% A; 0.25% min ^-1 ). 86 mg of pure product were obtained.

Ac-Leu-Ala-Asn-Gly-Val-Glu-NH-CH [CH ₂ CH (CH ₃ ) ₂ ] -CH ₂ -Arg-Asp-Asn-Gln-Leu-NH ₂

0.8 g resin according to Rink et a. (Fa. NOVABIOCHEM) corresponding to a batch size of 0.4 mmol was in accordance with 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 the Fmoc protective group had been removed (steps 2 to 4 were carried out according to Alb), the resin was suspended in DMF containing 1% acetic acid, 3 equivalents of Fmoc-leucinal (produced from Fmoc-leucine O, N-dimethylhydroxamate according to Castro, B ., Fehrentz, J.-A. (1983) Synthesis, 676-678) and 3 equivalents of NaBH ₃ CN. After 3 h (negative ninhydrin test), washing was carried out intensively and according to Alb with 1 mmol each

Fmoc-Glu (OtBu) -OH

Fmoc-Val-OH Fmoc-Asn (Trt)

Fmoc-Gly-OH Fmoc-Ala-OH

Fmoc-Leu-OH implemented. When synthesis was complete, the N-terminus

acetylated (execution of steps 2 to 4 and 8 to 9 according to Alb). The peptide resin was dried in vacuo (yield:

1.55 g).

Claims

1. Compounds of formula I.

()

X

wherein

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

or

- (CH ₂ ) _b -T

(with b meaning a number from 1 to 6 and T meaning OH-, CH ₃ S-, (CH ₃ ) ₂ CH-, C ₆ H ₅ -, p-HO-C ₆ H ₄ -, HS-, HO-CO-,

H ₂ N-CO, H ₂ N or H ₂ NC (= NH) -NH group) mean

F means Asp, Asn or His,

T ¹ -T ^{6 are} hydrogen atoms or methyl groups,

Z ¹ -Z ⁶ each for one oxygen atom or two

Hydrogen atoms stand

X the groups G-, G-NH-CHM-CO-, G-NH-CHM-CO-W-,

G-R-NH-CHM-CO- or G-R-NH-CHM-CO-W- means and Y -P, -NH-CHQ-CO-P, -V-NH-CHQ-CO-P,

-NH-CHQ-CO-U-P, -V-NH-CHQ-CO-U-P,

-NH-CHQ-K-NH-CHL-CO-P,

-V-NH-CHQ-K-NH-CHL-CO-P,

-NH-CHQ-K-NH-CHL-CO-U-P or

-V-NH-CHQ-K-NH-CHL-CO-UP, where in X and Y

G is a hydrogen atom or a

Amino protecting group means

P represents an OH or NH ₂ group or a carboxyl protecting group or

G and P together also mean a covalent bond or the group -CO- (CH ₂ ) _a -NH-, where a is a number from 1 to 12,

K, R, U, V and W peptide chains from 1-5

represent naturally occurring α-amino acids,

L, M and Q are hydrogen atoms or one of the groups -CH ₃ , -CH ₂ -CH (CH ₃ ) ₂ ,

-CH ₂ -CO-NH ₂ , - (CH ₂ ) ₃ -NH-C (NH) -NH ₂ , - (CH ₂ ) ₂ -CO-NH ₂ , -CH (CH ₃ ) ₂ , -CH ₂ OH ,

- (CH ₂ ) ₂ COOH, are or

A, B, D, M, L and Q in pairs one or two - (CH ₂ ) _c -SS- (CH ₂ ) _d -,

- (CH ₂ ) _e -CO-NH- (CH ₂ ) _f - or

- (CH ₂ ) _e -NH-CO- (CH ₂ ) _g -NH-CO- (CH ₂ ) _f bridge (s) (with c and d meaning a number from 1 to 4, e and f one Number from 1 to 6 and g of a number from 1 to 12), and their salts with physiologically tolerated acids.

2. 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 A, B, D, M, L and Q are not connected to one another.

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 - (CH ₂ ) _c - SS- (CH ₂ ) _d - bridge.

4. 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 group

- (CH ₂ ) _e -NH-CO- (CH ₂ ) _f - or - (CH ₂ ) _e -NH-CO- (CH ₂ ) _g -NH-CO- (CH ₂ ) _f .

5. Peptides according to claim 1, wherein G + P together a

covalent bond or -CO- (CH ₂ ) _a -NH- mean.

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 - (CH ₂ ) _c - SS- (CH ₂ ) _d - or - (CH ₂ ) _e -NH-CO- (CH ₂ ) _f bridges (with e and f in the

The meaning of a number from 1 to 4).

7. 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 Z ¹ to Z ⁶ each represent two hydrogen atoms.

8. Peptides according to claim 1 to 7 for use in the

Fighting disease.

9. Use of the peptides according to claims 1 to 7 for combating neoplastic diseases and autoimmune diseases and for combating and preventing

Infections, inflammation and rejection in transplants.

10. A process for the preparation of the peptides according to claim 1 to 7, characterized in that they are prepared by methods known in peptide chemistry.