CA2005060A1 - Tnf peptides - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE:

Peptides of the formula X-A-B-Tyr-Y, where A, B, X and Y are defined in the description, and the preparation thereof are described. The novel peptides are suitable for controlling diseases.

Description

2005(~0 O. Z . 0050/40386 NOYEI. TNF PEPTIDES

The pre~ent in~ention relates to novel peptide~ derived from tumor necrosis factor (TNF), the preparation thereof and the u~e thereof as drugs.

Carswell et al. (Proc. Natl. Acad. Sci. USA 72 (1975) 3666) reported that the ~erum of endotoxin-treated animals which had previously been infected w~th the Calmette-Guerin ~train of Mycobacteria (8CG) brought about hemorrhagic nscro~is in variou~ mou~e tumors. Thi3 activity was ascribed ~o tumor necrosis factor. TNP also has a cytostatic or cytotoxi~ effect on a l~rgs number of transformed cell line~ in vitro, whereas normal human and animal cell lineR are unaffected (~ymphokine Report~ Vol.

2, pp 235-275, Academic Pre~, Naw York, 1981). Rec~ntly, lS the biochsmical characterization and the gene for human TNF have bean described (Nature 312 (1984) 724; J. Biol.
Che~. 260 (1985) 2345; Nucl. Acids R~s. 13 (1985) 6361).

It is po~sible to deduce from thi~ data the followLng protein structure for mature hum~n TNFs V ~ y ~ Val~k~sValV~Li~A~DBo G~
Val~ ~~ v~lv~
GonV~LJa~teLy~GlyG~$~ ~ V l~
~Ed~gI~V~ q5rGbfnlLysV~U~ U~ leLys~ o Cy G~YbqC1urhJ~sG~uGlyA~ lAl PT y~rp~nCluProIld~nieu ~ I1sI1s~Al.qT~

The qN~ gene~ of catt1e~, rabbits and mice have al~o been de~cribed ( Cold Spring Harbor Symp . Quant . ~iol . 51 ( 1986 ) 597 ) .

Be~ides it~ cytotoxic propertie~, ~F i~ ona of the main 20C~5060 - 2 - O.Z. 0050/40386 substances involved in inflammatory reactions (Pharmac.
Re~. 5 (1988) 129). Animal models have shown that TNF i~
involved in 3eptic ~hock (Science 229 (1985) 869) and graft-ver~us-host diseas2 (J. Exp. Ned. 166 (1987) 1280).

We have now found that peptide~ with a considerably lower molecular weight have benefical propertie~.

The present invention relate~ tspeptide~ of the formula I

~-A-B-Tyr-Y I, where 10 A i~ Ser, Val, Ile or Pro, B is Gln or Ser, X is G-NH-CHM-CO-, G-NH-CHM-CO-W-, G-R-NH-CHN-CO- or -G-R-NH-CHM-CO-W- and Y is -Z, -NH-CHQ-CO-Z, -V-NH-CHQ-CO-Z, -NH-CHQ-CO-~-Z
or -V-NH-CHQ-CO-U-~
where, in X and Y, G is hydrog~n or an amino-protective group, Z i~ OH or NH2 or a carboxyl-protective group or G and Z togather are also a covalent bond or -CO-(CH2),-NN- where ~ i~ from 1 to 12, R, U, V and W are peptide chain~ compo~ed of 1-4 n~tur lly occurring c-a~ino acids, and N and Q are hydrogens or one of th~ following -C~(CH3)2, -CH~ C~3 )-C2H5, -C~H~, -CH(OH)-CH3, 2 5 --CH ~ --CH 2~ ( CH 2 ) b--T
H H
(with b being from 1 to 6 and T baing hydrogen or OH, CHaO, CB3S, (CH3)2CH, C~H5, p-HO-C~H~, HS, H2N, HO-CO, H2N-CO or H2N-C~=N~)-NH) or ~ ~nd Q together are a -(CH2)o~S~S~~CH2)t~~ -(CH2)o-CO~NH~
(CH2)r~ or -tcH2).-NH-co-(cH23~-NH-co-(cH2)~- bridge (with c and d be~ng from 1 to 4, e and f b4ing from 1 to 6 and g b6ing fro~ 1 to 12), - 3 - O.Z. 0050/40386 as well as the salt~ theraof with physiologically toler-ated acid~.

The peptide~ of the formula I are constructed of L-amino acid~, but they can contain 1 or 2 D-amino acid~. The S side-chains of the trifunctional amino acids can carry protective group~ or be unprotected.

Particularly preferred phy~iologically tolerated acid~
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, ascor-bic acid and acetylglycine.

~he novel peptide~ can be open-chain (G = H, amino-protective group; Z = OH, NH2~ carboxyl-protective group, M ant Q not connected together) and, in particular, have a di~ulfide bridge (G = H, amino-protective group;
Z = OH, NH2~ carboxyl-protective group; M ~ Q = -(CH2)C-S-S-(CH2)d-) or a side-chain bridge (G ~ H, amino-protec-ti~e group, Z - OH, NH2~ carboxyl-protective group, M + Q
5 - ( CH2 ) ~-~H-CO- (CH2)~- or -(CH2).-~H-CO-(CH2)6-NH-CO-(CH2)s-) or be linked head-to-tail (G ~ Z = covalent bond or _CO-~cH2).-N~

~he novel compounds can be prepared by conventional methods of peptide chemi~try.

Thu~, the peptides can be constructsd ~eguentially from amino acids or by linking together 3uitable ~m~ller pept$de fragment~. In the sequential construction, the pep~id~ chain i~ axtended stepwise, by one amino acid each tim~, starting at the C ter~inu~. In the case of fragment coupling, it is po~sible to link together ZC~05060 4 - O.Z. 0050/40386 fragments of different lengths, these in turn being obtainable by sequential conctruction from amino acids or coupling of other fragment~. The cyclic peptides are obtained, after ~ynthesis of the open-chain peptides, by a cyclization reaction c~rried out in high dilution.

In the ca~e both of sequential construction and of fragment couplîng, it i8 neces~ary for the building blocks to be linked by formation of an amide linkage.
Enzymatic and chemical methods are suitable for thi~.

Chemical method~ for forming amide linkageY are dealt with in detail by Muller, Nethoden der Organi~chen Chemie (Nethods of Organic Chemistry) Yol. XV/2, pp 1-364 r Thieme Verlag, Stuttgart, 1974; Stewart, Young, Solid Phase Peptide Synthe~is, pp 31-34, 71 82, Pierce Chemical Company, Rockford, 1984; Bodanszky, ~lausner, Ondetti, Peptide Synthe~is, pp 85-128, John Wiley & Sons, New York, 1976 and other standard works of peptide chemistry.
Particularly preferred are the azide method, the sy~metr-ical and mixed anhydrid~ method, active esters generated in situ or pr~formed and ths formation of amide linkage~
using coupling reagents (activator~), in particular dicyclohexylcArbodiimide (DCC), diisopropylcarbodiimide (DIC), l-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (E~DQ), l-ethyl-3-(3-dimethylaminopropyl)carbodiLmide hydrochlor~de (~DCI), n-propanephosphonic anhydride ( PPA), N , N-bi~ (2-oxo-3-oxazolidinyl)amidophosphoryl chloride (BOP-Cl), diphenylpho~phoryl azide (DPPA), Ca~tro'~ r0sgent (BOP), O-benzotriazolyl-N,N,N',N'-tetra-methyluronium ~alts (H~TU), 2,5-diphenyl-2,3-dihydro-3-oxo-4-hydroxythiophe~e dioxid~ (Staglich~s reagent;
HOTDO) a~d l,1'-carbonyldiimidazole (CDI). The coupling reagents can be ~mployed alone or in combination with additives ~uch as N,N'-dimethyl-4-aminopyridine (DMAP), ~-hydroxybensotriazole (HOBt~, N-hydroxybanzotriazine 3S (HOOBt), N-hydroxy~uccinimidQ (HOSu) or 2-hydroxy-5 - O.Z. 0050/40386 pyridine.

Wherea~ it is normally possible to dispen~e with protective groups in enzymatic peptide ~ynthe~is, for chemical ~ynthesi~ it is nece~sary for there to be reversible protection of the reactive functional groups which are not involved in the formation of the amide linkage on the two reactant~. Three conventional pro-tective group techniques are preferred for chemical peptide ~yntheses: the benzyloxycarbonyl (Z), the t-butyloxycarbonyl (Boc) and the 9-fluorenylmethyloxy-car~onyl (Fmoc) technique~. In each ca~e the protecti~e group on the ~-~mino group of the chain-extending build-ing block i~ identified. The side-chain protective groups on the trifunctional amino acid~ are chosen ~o that th~y are not nece~sarily eliminated togethar with the ~-amino protectivs group. A detailed review of amino acid protec-tive group~ is given by Muller, Methoden der Organischen Che~ie Vol XV/l, pp 20-906, Thieme Verlag, Stuttgart, 1974.

The building blocks u~ed to construct the peptide chain can be reactet in ~olution, in suspension or by a msthod ~imilar to that de~cribed by Nerrifield in J. Amer. Che~.
Soc. 85 (1963) 2149. Particularly preferred methods are those in which peptides are constructed sequ2ntially or by fragment coupling by u8e of the Z, Boc or Fmoc protec-tiVQ group technique, in which ca~e the reaction takes place in ~olution, as well as those in which, ~imilar to the ~errifield technique, one reactant i8 bound to an in~oluble polymer$c support (al~o called re~in herein-after). This typically entails the peptide being con-structed sequentially o~ the poly~eric ~upport, by u~e of the Boc or Fmoc protective group technique, with the growing peptide chain being covalently bonded at the C ter~inua to the insoluble resin particle~ (cf. Figure~
35 1 and 2 ) . This procedure allow~ reaqents and byproduct~

~ 6 - 0.2. 0050/40386 to be removed by filtration, and thus recry~tallization of intermediate~ uperfluous.

The protected amino acid~ can be bonded to any suitable polym~rs which merely need to be insoluble in ths 801-vents used and to have a stable phy~ical form whichallows easy fîltration. The polymer must contain a functional group to which the first protected amino acid can be firmly linked by a covalent bond. A wide variety of polymers is suitable for thi~ purpose, for example cellulose, polyvinyl alcohol, polymethacrylate, sulfon-ated polystyrene, chloromethylated copolymer of styrene and divinylbenzene (Merrifield resin), 4-methylben~-hydrylamine-resin (MB~A-re~in), phenylacetamidomethyl-resin (Pam-resin), p-benzyloxybsnzyl alcohol-resin, benzhydrylamine-re~in (BHA-resin), 4-hydroxymethyl-benzoyloxymethyl-resin, the re~in u~ed by Breipohl et al.
(Tetrahedron Lett. 28 (1987) 565; from BACHE~), HYCRAM
re~in ~from ORPEGEN) or SASRIN resin (from ~ACHEM).

Solvents ~uitable for peptide ~ynthesis in ~olution are all those which are inert under the reaction conditions, in particular water, N,N-dimethylformamide (DNF), dimethyl sulfoxide (DNSO), acetonitrile, dichloromethane (DCM), 1,4-dioxana, tetrahydrofuran (THF), N-methyl-2-pyrrolidone (NMP) and ~ixture~ of the said ~olvent~.
Peptide synthesis on polymeric supports ~an be carriad out in all inart organic solvents which dissolve the amino acid derivative~ u~ed; however, solvent which also have resin-sw~lling propertie~ are preferred, such as DMF, DCM, NNP, acetoni~rile and D~SO, a~ well a~ mixture~
of tha~e ~olvent~.

After the peptide ha~ been ~ynthesized it i~ cleaved off the polymeric ~upport. The clea~age conditiona for the variou~ types of r2sin~ are disclosed in the literature.
The cl~avage reactions most commonly use acid and 2C~05060 - 7 - O.Z. 0050/40386 palladium catalysi~, in particular cleavage in anhydrous liquid hydrogen fluoride, in anhydrous trifluoromethane-sulfonic acid, in dilute or concentrated trifluoroacetic acid or palladium-catalyzed cleavage Ln THF or THE-DCM
mixtures in the pre3ence of a weak base ~uch as morpho-line. The protective groups may, depending on the choice thereof, be retained or likewise cleaved off under the cleavage conditions. Partial deprotection of the peptide may al~o be worthwhile if the intention i~ to carry out certain derivatization reactions or a cyclization.

Some of the novel peptides have good cytotoxic proper-ties. Som~ other~ of the peptide~ have high affinity for the cellular TNF receptor without, however, having cytotoxic activity. They are therefore TNF antagoni~t~.
They compete with natural ~NF for binding to the cellular TNF receptor and thus suppress the TWF effect. The novel peptides are valuable drug~ which can be employed for treating neopla~tic di~Qase~ and autoimmuns disease~ as well a~ for controlling and preventing infection~, inflammations and transplant re~ection reaction~. Simple expsriment~ can be u~ed to alucidate the mode of action of the individual peptides. The cy~otoxicity of the peptide i~ determined by incubating a TNF-sensitive cell line in the pre~ence of the peptide. In a second experi-mental approach, the cell line is incubated with therelevant peptide in the presence of a lethal amount of ~NP. It iB possibla in this way to detect the TNF-ant~goni~tic effect. In addition, the affinity of the peptide for the cellular TNP receptor i8 determined in an in vitro binding experiment.

~he following test ~ystems were used to characterize the agonistic and antagonistic effect~ of th~ novel p~ptides:

I. Cytotoxicity t2st on TNF-sen~itive indicator cells, I~. Cytotoxicity antagonis~ t~ on TN~-~ensiti~e - 8 - O.Z. 0050/40386 indicator cells, III. Competitive receptor-binding test on indicator cell~
expressing TNF receptor.
I. Cytotoxicity test The agonistic effects of the novel peptide~ are as~essed on the ba~i~ of their cytotoxic effect on TNF-sensitive cells (e.g. L929, MCF-7, A204, U937).
The test with L929 and MCF-7 was carried out a~
follows~

1. 100 ~1 of culture medium containing 3 to 5 x 103 fre~hly trypsinized, exponentially growing, L929 cells (mou~e) or ~CF-7 cells (human) were pipetted into the well8 of a 96-well flat-bottom culture plate. The plate was incubated at 37C
overnight. The air in the $ncubator wa~ saturated with water vapor and contained 5% COz by volume.

The L929 culture medium contained 500 ml of lx Earle's NEM (Boehringer Mannheim)~ 50 ml of heat-inactivated (56C, 30 min) fatal calf serum (PCS), 50 ml of L-glutamine (200 mM), 5 ml of lOOx non-essentLal amino acids, 3 ml of lM HEPES
buffer pH 7.2,and 50 ml of gentAmicin (50 mg/ml).

Th~ NCF-7 culture mediu~ contained 500 ml of lx Dulbecco'~ MEM (~oehringor Mannheim), 100 ml of heat inactivated (56C, 30 min) FCS, 5 ml of L-glut~mine and 5 ml of lOOx non-e~sential amino acids .

2. The next day 100 ~1 of the peptide solution to be te~ted were added to the cell cultures and ~ub~ected to serial 2-fold dilution. In addition, some cell control~ (i.e. cell cultures not treated with peptide dilutio~) and ~o~e rhu-TNF
control~ (i.e. cell cultures treated with recom-2()05060 g o.Z. 0050/40386 binant human TNF) were al~o made up. The culture plate wa~ incubated at 37C in an atmosphere of air saturated with water vapor and containing 53 C02 by volume for 48 h.

3. The percentage of surviving cell~ in the cultures treated with peptide dilution wa~ determined by ~taining with cry~tal violet. For thi~ purpo~e, the liquid wa3 removed from the wells of the test plate by tapping it. 50 ~1 of crystal violet solution were pipe~ted into each well.

- The composition of the crystal violet solution wa~ as followss 3.75 g of cry3tal violet 1.75 g of NaCl 161.5 ml of ethanol 43.2 ml of 37% formaldehyde water ad 500 ml The crystal violet ~olution wa~ left in the well~
for 20 min and then likewise removed by tapping.
The plates w~re then w~hed 5 time~ by immar~ion in water in order to remove dye not bound to the cell~. The dye bound to the cell~ wa~ extracted by ~dding 100 ~1 of ra~gent solution (50% etha-nol, 0.1% glacial acetic acid, 49.9% water~ ~o each well.

4. Th~ pl~tes were shaken for 5 min to obtain a solution of un~form color in each well. The surviving cell~ w~re determined by mea~uring the extinction at 540 nm of tho colored ~olution in the individual wells.

5. Subsequently, by relating to the cell control, Z1~05060 - 10 - O.Z. 0050/403~6 the 50~ cytotoxicity value was defined, and the reciprocal of the sample dilution which requlted in 50~ cytotoxicity was calculated as the cyto-toxic activity of the te~t 8ample.

II. Cytotoxicity antagonism te~t The antagonistic effect of the peptides was assessed on the ba3is of their property of anta~onizing the cytotoxic effect of rhu-TNF on TNP-sen~itive cell3 (Q.g. L929, MCP-7, A204, U937). The cytotoxicity antagoni~m test with L929 and NCF-7 cell~ was carried out as follows:
1. 100 ~1 of culture medium containing 3 to 5 x 103 freQhly trypsinized, exponentially growing, L929 cells (mou3e) or ~CF-7 cell8 (human) were pipetted into the wells of a 96-w~ll flat-bottom culture plate. The plate wa~ incubated at 37C
overnight. The air in the incubator was saturated with water vapor and contained 5% CO2 by volume.

T~e L929 culture medium contained 500 ml of lx Earle'~ MEM (Bo~hringer Mhnnheim), 50 ml of heat-inactivated (56C, 30 min) FCS, 5 ml of L-gluta-mine (200 m~), 5 ml of lOOx non-es~ential amino acld~, 3 ml of lN B PES buffer pH 7.2, and 500 ~1 of gent~micin (50 mg/ml).

Th~ ~CF-7 culture medium cont~ined 500 ml of lx Dulbecco~s MEM (Boehringer ~nnnheim), 100 ml of heat in~çtiYated (56-C, 30 min) FCS, 5 ml of L-glutamine (200 m~) and 5 ml of 100~ non-essential amino acids.

2. The nex~ day lO0 ~1 of the peptide ~olution to be te~ted were added to the cell cultures and ~ub~ected to ~erial 2-fold dilut~on. Then, 100 ~1 of a rhu-TNP dilution in culture mediu~, which 20050~i0 ~ O.Z. 0050/4038b dilution had an 80-100% cytotoxic effect in the final concentration in the cell culture, were added to these cell culture~. In addition, ~ome cell control~ (i.e. cell culture~ not treated with peptide ~olution or with rhu-TNF ~olution) and some rhu-TNF control~ (= cell cultures treated only wi~h rhu-~NF ~olution) were also made up. The culture pla~e wa~ then incubated at 37C in an atmosphere of air saturated with water vapor and containing 5% CO2 by vol~me for 48 h.

3. The percentage of ~urviving cell~ in the cultures treated with ~ub~tance solution was determined by ~taining with cry~tal violet. For thi~ purpose, the liquid was removed from tha well~ of the test plate by tapping it. 50 ~1 of crystal violet solution were pipetted into each well.

The cry~tal violet solution had the compo~ition specified in I.3 The crystal violet solution waH left in the well for 20 ~in and then likewise re~oved by tapping.
The plate~ were then washed 5 ti~e~ by immersion in water in order to r~move dye not bound to the cell~. The dye bound to the cells was extracted by adding 100 ~1 of reagent solution (50~ etha-nol, 0.1~ glacial acet$c acid, 49.9S waterJ to each well.

4. The plate~ were shaken for 5 min to obtain a ~olution of uniform color in eAch wall. The ~urviving cells were determined by measuring the extinction at 540 nm of the colored solution in the individual well8.

5. Subsequently, by relating to the cell control and - 12 - O.Z. 0050/40386 the rhu-TNF control, the 50% antagoni~m value ~as defined, and the ~ample concentration which resulted in 50% antagoniqm of rhu-T~F
cytotoxicity at the rhu-TNF concent-ation used wa~ calculated as antagonistic ac~ivity of the sample te~ted.

III. Competitive receptor-binding te~t Both the agonistic and antagonistic effects of peptide~ are conditional on the latter binding to the ~NF raceptor. Thi~ means that peptides with an agoni~tic or antagoni~tic effect compete with rhu-TNF for binding to the TNF rec~ptor on TNF-q~n~itive indicator cells (e.g. U937). The ~ompeti-tive receptor-binding te~t was carried out a~
followss 1. 100 ~1 of medium containing various concentra-tion~ of the peptide to be te~ted and of rhu-TNF
(= control) were pipetted into the reaction ve~sels. The medium compri~ed 500 ml of PBS
(Boehringer Msnnheim), 10 ml of he~t-inactivated ~56C, 30 min) PCS and 100 mg of ~odium azide.

2. Subsequently, 100 ~1 of medium cont~ining 1 ng of ~ labeled rhu-~NP (Bolton lactoperoxida~e method) were placed in the reaction VQE8elB and mixed. The non-spscific binding (NSB) was deter-~lned by mixing in the reaction ve~sel~ the ~5I-labQled rhu-TNP (1 ng of ~'I-rhu-TNF in 100 ~1 of med~um) with a 200-fold e~co~s of unlabeled rhu-TNF (200 ng of rhu-TNF in 100 ~1 of medium).

3. Then 100 ~1 of medium containing 2 x 10~ U937 cells (human) were pipetted into the raaction v~el~ and mixed. The rea~tion ve~891E (test volu~e 300 ~1) were incubated at 0C for 90 min.

2C~OS060 - 13 - O.Z. 0050/40386 The reaction mixture~ were remixed after 45 min.

4. After the incubation, the cell~ were centrifuged at 1800 rpm and 4C for 5 min, washed 3 time8 with medium and transferred quantitatively into S counting vials, and the cell-bound radioactivity was determined in a Clini gamma counter 1272 lI,RB ~allac).

5. After the measurement~ had been corrected for the non-~pecific b~nding, the 50~ compstition value 0 wa8 defined by relating to the overall binding, and the sample concentration which led to 50~
competition of ~5I-rhu-TNF binding at the125I-rhu-~NF concentration u~ed was calculated as the competitive activity of the sampla tested.

~he Examples which follow are intended to explain the in~ention in more detail. The proteinogenous amino acids are abbreviated in the Example~ u~ing the conYentional three-letter code. Other meaning~ are~
Ab~ = 4-aminobutyric acid, Ac - acetic acid, Bal - ~-al~niné, Hcy = homocysteine, Hly = homolysine, Orn = ornithine, Dap = 2,3-diaminopropionic acid.

A. Gsneral procedures I. The peptide~ claimed in claim 1 were ~ynthesized u~ing standard me~hod~ of solid-phase peptidQ
~ynthssis in a completely automatic modal 430A
p~ptide synthe~izer from APPLIE~ BIOSYSTE~S. The apparatu~ u~es different synthe~is cycle~ for the Boc and Fmoc protecti~e group technique~.

a) Synthe~is cycla for the Boc protective group technique 1. 30~ ~flUoDU~eia acid in DCM 1 x 3 min 2iDOS060 - 14 - O. Z .0050/40386 2. 50% triflu~cetic a~:id in DCII 1 x 17 min 3. DCM ~sl~ g 5 x 1 min 4. 596 dli~l~t}~lam~ in DCM 1 x 1 min 5. 59~ dll~let~lamine in ~P 1 x 1 min 6. I~P wa~hi~ 5 x 1 min 7. h~iti~n of F~tivat~l p~tect~ amin~
a~id (a~tivation }~y 1 ~ival~nt of D~
arxl 1 eqpival~c of DBt in NN~/~);
pepti.de calpl.~ (~ part) 1 x 30 min 8. ~diti~n of I~MSO to th~ reacti~ mixl~re l it contain~ 2096 nt5so ~y volu~

9. Pep~.da c~upling (2~ part)1 x 16 min 10. Ad~iti~ of 3.8 eq~ivaler~ of dii~

11~ PeQtide wpling (3~ prt) 1 x 7 min 12. DC:M ~hing 3 x 1 min of cc~ling (se~ to S.) 14 . 10% aoe~ ar~ide, 5~ d~l ~ ,lamine in DCM 1 x 2 min 15. 10% acetic ar~ride in DOS1 x 4 min 16. ~QI washing 4 x 1 min 17 ~3tum to 1.

1. NIP wo~ing 1 x 1 Inin 2. 20% p~p~id~ in N~P 1 x 4 min 3. 20% p~id~ NMP 1 x 16 mi~l 4. Nt~P wa~ling S x 1 min 5. A~diti~l of pn3a~tivated E~ ~millo a~id (activati~xl ~y 1 ~val~t of D~C

pç~ptide a~upling 1 x 61 min 6. ~qP ~hir~ 3 x 1 min 7. If re~ctian i~ ir~te, r~0titi~ of ca~lin~ (r~ to 5. ) 8. 10% ~c a~ydr~e in ~? 1 x 8 min - 15 - o- 7~ 0050/40386 9. NMP washing 3 x 1 min 10. R~hLn to 2.

II. Working up of peptide-resins obtained as in Ia The peptide-resin obtained as in Ia wa~ dried under reduced pressure and tran~ferred into a reaction ve~el of a Teflon HF apparatu3 (from PENINSULA).
Addition of a scavenger, preferably ani~ole (1 ml/g of re~in), and of a thiol in the ca~e of tryptophan-containing peptides, to remove the indole formyl group, preferably ethanedithiol (0.5 ml/g of resin), was followed by conden~ation in of hydrogen fluoride (10 ml/g of re in) while cooling with liquid N2. The mixture was allowed to warm to O-C, and was stirred at thi~ temperature for 45 min. The hydrogen fluor-ide wa~ then ~tripped off under reduced pressure and the residue wa~ wa~hed with ethyl acetate in order to remove remaining sc~venger. The peptide was axtract~d with 30~ ~trength acetic acid and filtered, and the filtrate was fre~ze-dried.

~o prepare peptide hydrazides, the peptide-re~in (Pam- or N~rrifield resin) wa~ suspended in DME
(15 ml/g of resin), hydrazinQ hydrate (20 equiva-lent~) was added, and the mixtu~e wa~ ~tirred at room tRmperature for 2 days. To work up, the reain wa~ f~ltered off and the filtrate wae evaporated to dryne~. Th2 residue wa~ crystallized from DNP/~t20 or MeOHl~t2O.

III. Working up of the peptide-res~ns obtained a~ in Ib The peptid~-ra6in obtainffd as in Ib was dried u~der reduced pressure and sub~equently ~ub~ected to one of the following cleav~ge procedurss, depending on the amino acid compo~ition (W~de, Tregear, Howard Florey Fmoc-Work~hop Nanual, Nelbourne 1985).

- 16 - O.Z. 0050/40386 Peptide containing Cleavage conditions _ _ Arq(Mtr) ~et Trp TFA Scavenger Reaction time S
no no no 95~ 5~ H20 1.5 h yes no no 95% 5% thioanisole ~ 3 h no ye~ no 95~ 5% ethyl methyl 1.5 h 9ul fide no no yes 9S% 5% sthanedithiol/1.5 h anisole (1:3) no ye~ yes 95~ 5~ ethanedithiol~l.s h ani~ole/ethyl methyl sulfide (1:3:1) ye3 ye~ yes 93% 7% ethanedithiol/~ 3 h ani~ole/ethyl methyl ~ulfide (1:3:3) ~he ~u~pension of the peptide-re~in in the suitable ~FA mixture wa~ stirred at roo~ temperature for the ~tated time and then the re~in wa~ filtered off and wa~hed with TFA and with DCN. The ~iltrate and the washing~ were exten~ively concentrated, and the peptide wa~ precipitated by addition of diethyl ~ther. The mixture was cooled in an ice bath, and th~ pr0cipitate wa~ filtered off, taken up in 30%
acetic acid and freeze-dried.

IV. Purification and characteriz~tion of the peptide~

Purification was by gel chromatography (SBPHADE~
G-10, G-15/10% HQAc; SEPHADEXO LH20/MeOH) and ~ub-sequent medium pre~sure chromatogr~ph~ (stationary phase~ HD-SIL C-18, 20-45 ~, 100 A; mobile phase~
gradiant with A = 0.1% TFA/MeOH, ~ - 0.1% TFA/H20).

The purity of the final products was determined by andlytical HPLC (sta~ion~ry pha~es 100 x 2.1 mm Z()05060 - 17 - O.Z. 0050/403~6 VYDAC C-18, 5 ~, 300 A; mobile pha~e = CH3CN/H20 gradient buffered with 0.1% TFA, 40C). Charac-terization was by means of amino acid analy~i~ and fast atom bombardment mass spectro~copy.

B. Specific procedure3 EXAMPL~ 1 H-Arg-Ile-Ala-Val-Ser-Tyr-Gln-Thr-Lys -NH2 1.38g of Boc-Lys(C1-Z)-p-~BHA-resin (~ubstitution 0.36 mmol/g), corresponding to a batch size of 0.5 mmol, were reacted as in AIa with 2 m~ol each of Boc-Thr(Bzl)-OH Boc-Val-OH
Boc-Gln-OH Boc-Al~-OH
Boc-Tyr(Br-Z)-OH Boc-Ile-OH
Boc-S~r(Bzl)-OH Boc-Arg(To~)-OH

After the 3ynthesis was complete, the peptide-re~in underwent N-terminal deprotection (stepa 1-3 a~ in AIa) and ~ubsequent drying under reduced pres~ure; the yield wa~ 2.3 g.

lolS g of th~ resin obtained in thl~ way were sub~ected to HF cleavage a~ in AII. The crude product (195 g) wa~
purified by gol filtration (SEPHAD~X G-10) and medium pres~ure chromAtography (cf. ~IV; 60-75 ~ A; 0.25 ~ min~l).
102 ~g of pure product were obtained.

EXAMP~ 2 Ac-Ile-Ala-Val-Ser-Tyr-Gln-Thr-OH

0.4 g of Fmoc-Thr(t-Bu)-p-alkoxybenzyl alcohol-re~in (~ubstitution 0.63 mmol/g), correRponding to a batch size of 0.25 ol, was reacted.a~ in A~b with 1 mmol each of Fmoc-Gln-OH Fmoc-Val-OH

- 18 - O.Z. 0050/40386 Fmoc-Tyr(tBu)-oH Fmoc-Ala-OH
Fmoc-Ser(tBU)-OH Fmoc-Ile-OH

After the synthe~is wa~ complete, the N tQrminus was acetylated (step~ 2-4 and 8-9 as in AIb). The resulting peptide-reYin was dried under reduced pre~ure; the yield wa~ 0.6 g.

The crude peptide (160 mg) obtained after TFA cleavage ?~
in AIII was purified by gel filtration (5EPHADEX~ G - 10) and medium pressure chromatography (cf. AIV; 60-75 ~ A;
0.25 % min~l). 87 mg of pure product were obtained.

The following can be prepared in a ~imilar manner to Example~ 1 and 2:
3. H-Ala-Val-Ser-Tyr-Gln-OH
4. Ac-Ala-Val-Ser-Tyr-Gln-OH
5 H-Ala-Val-Ser-Tyr-Gln-NH2 6. Ac-Ala-Val-Ser-Tyr-Gln-NH2 7. H-Ala-Val-Gln-Tyr-Gln-OH
8. Ac-Ala-Val-Gln-Tyr-Gln-OH
9. H-Ala-Val-Gln-Tyr-Gln-NH2 10. Ac-Ala-Val-Gln-Tyr-Gln-NH2 11. H-Ile-Ala-Val-Ser-Tyr-Gln-OH
12. Ac-Ile-Ala-Val-Ser-Tyr-Gln-OH

13. H-Ile-Ala-Val-Ssr-Tyr-Gln-NH2 14. Ac-Ile-Ala-Yal-Sar-Tyr-Gln-NH2 15. H-Phe-Ala-Val-Ser-Tyr-Gln-OH

16. As-Phe-Ala-Val-Ser-Tyr-Gln-OH

17. H-Phe-Ala-Val-Ser-Tyr-Gln-NH2 18. As-Phe-Ala-Val-Ser-Tyr-Gln-NH2 19. ~-Ile-Ala-Val-Ser-Tyr-Gln-Thr-OH

20. Ac-Leu-Ala-Val-Ser-Tyr-Gln-Thr-OH

21. H-Ile-Ala-Val-Ser-Tyr-Gln-Thr-NH2 22. Ac-Ile-Ala-Val-Ser-Tyr-Gln-Thr-NH2 23. H-Phe-Ala-Val-Ser-Tyr-Gln-Thr-OH

24. ~c-Phe-Ala-Val-Ser-Tyr-Gln-Thr-OH

25. H-Phe-Ala-V l-Ser-Tyr-Gln-Thr-NH2 26. Ac-Phe-Ala-Val-Ser-Tyr-Gln-Thr-NH2 - 19 - O.Z. 0~50/4~386 27. H-Arg-Ile-Ala-Val-Ser-Tyr-Gln-Thr-Lys-OH

28. Ac-Arg-IlQ-Ala-Val-Ser-Tyr-Gln-Thr-Ly~-OH

29. H-Arg-Leu-Ala-Val-Ser-Tyr-Gln-Thr-Lys -NH2 30. Ac-Arg-Ile-Ala-Va1-Ser-Tyr-Gln-Thr-Lys-NH2 31. H-Ser Arg-Ile-Ala-Val-Ser-Tyr-Gln-lhr-Lys-Val-Asn~oH

32. Ac-Ser-Arg-Ile-Ala-Val-Ser-Tyr-Gln-Thr-Ly~-Val-A~n-CH

33. ~-Ser-Arg-Ile-Ala-Val-Ser-Tyr-Gln-lhr-Lys-Val-Asn-NH2 36. Ac-Ssr-Arg-Ile-Ala-Val-Ser-Tyr-Gln-lhr-Lys-V.l-Asn-NH2 37. H-Il~-Ala-Val-Ser-Tyr-Asn-OH
38. Ac-Ile-Ala-Pro-Ser-Tyr-Gln-Thr-NH2 39. H-Ile-Gly-Val-Ser-Tyr-Gln-Thr-OH
40. Ac-Arg-Ile-Ala-Val-Gln-Tyr-Gln-Thr-Ly~ -NH2 41. Ac~-Arg-Ile-Ala Val-SbrJTyr~Val-Lys-Val-Aan-NH2 H-Cy~-Ala-Val-Ser-Tyr-Cys -NH2 0.98g of Boc-Cys(pMB)-MBHA-resin (substitution 0.51 mmol/g), corresponding to a batch ~ize of 0.5 mmol, was reacted aa in AIa with 2 mmol each of Boc-Tyr(Br-Z)-OH Boc-Ala-OH
Boc-Ser(Bzl)-OH Boc-Cy8(pMB)-OH
Boc-Val-OH

~he peptide-re~in wa~ dried under reduced pre~sure; the yield was 1.5 g.

0.75 g of the resin obtained in this way was sub~ected to HF cleavaqe as in ~II. The freeze-dried c N de product wa3 taken up in 2 1 of 0.1% ~trength acetic acid, and the pH
wa~ then ad~ustad to 8.4 with aqueou~ ammonia. Under an argon atmo~phero, 0.01 N R3[Fe(CN)~3 solution was 910wly added dropwise until the yellowi~h-green color persisted for at lea~t 15 min. The mixtura wa~ ~hen stirred for 1 h and then ac$d~fied to pH 4.5 with glacial acetic acid, and 15 ml of an aqueous ~u~pen3ion of an anion exchanger (BIORAD 3 x 4A, chloride form) were added. After 30 min, - 20 - O.Z. 0050/40386 the ion exchanger re~in wa~ filtered off, and the filt-rat~ wa~ concentrated to 100 ml in a rotary evaporator and ~ubsequently freeze-dried.

All the solvents used had previously been ~aturated with nitrogen in order to prevent any oxidation of the free cysteine residues.

The crude product was purified by gel chromatography (SEPNALtEX~t G-15) and medium pre~sure chromatography (cf.
AIV; 20-40 % A; 0.25 % min1). 18 mg of pure product were obtained.

~he following can be prepared in a ~i~ilar manner to Example 42 (Pam-resin was used to prepare the peptide acids) ~

5 43. H-Cys-Val-Ser-Tyr-Cy~-OH

44. Ac-Cy~-Val-Ser-Tyr-Cys-NH2 j .
45. H-Cys-Val-Ser-Tyr-Cy~-NH2 46. Ac-Cys-Ala-D-Val-Ser-Tyr-Cys-NH2 47. N-Cy~-Ala-D-V~1-S~r-TyT-Cys-NH2 5 48. H-Cys-Val-Ser-Tyr-Gln-Cy~-NH2 t 49. As-Cys-Val-Ser-Tyr-Gln-Cy~-NX2 50. H-Cy~-Val-D-Ser-Tyr-Gln-Cys-NH2 ~ - I
51. As-Cy~-Val-D-Ser-Tyr-Gln-Cys-NH2 52. H-Cy~-Val-D-Ala-Tyr-Gln-Ci~-NH2 53. Ac-Cy~-Val-D-Ala-Tyr-Gln-Cy~-NH2 -- 21 - O.Z. 0050/40386 r 54. H-Cys-Ala-Val-Ser-Tyr-Cys-OH

55. Ac-Cys-Ala-Val-Ser-Tyr-Cy~-NH2 S
56. H-Hcy-Ala-Val-Ser-Tyr-Cy~-OH

57. Ac-Hcy-Ala-Val-Ser-Tyr-Cys-NH2 58. H-Hcy-Ala-Val-Ser-Tyr-Hcy-OH
r 59. Ac-Hcy-Ala-Val-Ser-Tyr-Hcy-NH2 60. H-Cys-Ala-Val-Ser-Tyr-Gln-Cy~-OH

61. H-Cys-Ala-Val-Ser-Tyr-Gln-Cys-NH~
I
62. Ac-Cy~-Ala-Val-Ser-Tyr-Gln-Cys-NH2 I
63. ~c-Cys-Gly-Val-Ser-Tyr-Gln-Cys-NH2 1' 64. H-Cy~-Ile-Ala-Val-Ser-Tyr-Gln-Cys-OH

65. Ac-Cys-Ile-Ala-Val-Ser-Tyr-Gln-Cy~ -NH2 1 _ I
66. H-Hcy-Ile-Ala-Val-Ser-Tyr-Gln-Cys-OH

67. H-Cys-Val-Gly-~yr-Gln-Cys-NH2 r 68. Ac-Cys-Val-Gly-Tyr-Gln-Cys-NH2 1- l 69. Ac-Hcy~ Ala-V41-Ser-Tyr-Gln-Cy~-NH2 A~-Cys-Val-Ser-Tyr-Gln-Thr-Ly~ -Cy8 -NH2 71. Ac-Cy~-Ala-Val-S~r-Tyr-Gln-Thr-Ly~-Cy~-N~2 72. ~-Cy~-Ile-Ala-Val-Ser-~yr-Gln-~hr-Lys -Cy8 -OX

`` - 22 - O.Z. 0050/40386 73. Ac-cy~ e-Ala-val-ser-Tyr~Gln-Thr-hys-cyq-NH2 74. Ac-Cy~-Arg-Ile-Ala-Val-Ser-Tyr-Gln-Thr-Ly~-Cys -NH2 75. H-Ile-Cys-Val-Ser-Tyr-Cys-Thr-OH

76. Ac-Ser-Arg-Cys-Ala-Val-Ser-Tyr-Cy~-Thr~Lys-NH2 77. A~-Hcy-Ala-Ile-Ser-Tyr-Cys-N~2 ~
78. H-Ile-Ser-Arg~-Ala-Val~JTyr-Gan~ Lys-Val-oH

79. Ac-Ile~br-Arg~Y-Ala-V~1~Ser~ Gln~-Ly~-Val-NH2 80. H-Cys-H~s~Thr-Ile-Ser-Arg-Ile_Ala-VAl~-Tyr-Gln-I
q~r-Ly~-Val-A~n-T~-Ieu-Ser-Ala~ys-NH2 81. Ac~-His-ISr-Ile~b~-Arg-Ile-Ala-Val~-lyr~n-Thr-Ly -~al-Asn-Ieu-Leu ~ r-Ala~-NH2 r Ac-Orn-Val-Ser-Tyr-Asp-NH2 0.53 g of Boc-Asp(OChx)-MBHA-re~in (~ubstitution 0.96 mmol/g), corresponding to a batch ~ize of 0.5 mmol, wa~
reacted a~ in AIa with 2 mmol aach of Boc-Tyr(Br-Z)-OH Boc-Val-OH
Boc-Ser(Bzl)-OH Boc-Orn(Z)-OH

Afte~ the synthe~i~ was complete, the N termin~s wa~
acetylated (BtQp~ 1-6 and 14-16 a~ in AIa). The resulting peptide-resin was dried under reduced pre~sure; the yield was 1.03 g.

250 mg of the crude product (305 mg) obtained after HF

., .

- 23 - O.Z. 0050~403a6 cleavage as in AII were di~solved in 350 ml of dega~sed DMF, and 0.3 ml of triethylamine and, at -25C, 0.3 ml of diphenylphosphoryl azide were added. The mixture was stirred at -25C for 2 h, ~tored at -25C for 2 h, at 4~C
for 2 days and at room temperature for 2 days and subse-quently evaporated to dryness. The crude pep~ide wa~
purified by gel chromatography (SEPHAD~ G-15). 51 mg of pure product were obtained.

~ I
Ac-Lys-Ala-Val-Ser-Tyr-Gln-A3p-NH2 1 g of resin described by Breipohl et al. (from BACHEM), corresponding to a batch size of 0.S mmol, was reacted as in AIb with 2 mmol each of Fmoc-Asp~OtBu)-OH Fmoc-Val-OH
Fmoc-Gln-OH Fmoc-Ala-OH
Fmoc-Tyr(tBu)-OH Fmoc-Ly~(Boc)-OH
Fmoc-Ser(tBu)-OH

After the synthesis was complete, the N-terminus was aceytlated (steps 2-4 and 8-9 as in AIb). The peptide-resin was dried under reduced pressure; yield 1.55 g.

The crude product (335 mg) obtained after TFA cleavage as in AIII was di~olved in 500 ~1 of daga~sed DMF, and 0.3 ml of tri~thylamine and, ~t -25~C, 0.3 ml of di-phenylphosphoryl a~ide were added. ~he mixture was stiredat -25-C for 2 h, stored at -25-C for 2 days, at 4cC for 2 days and at room temperature for 2 days and sub~equ~ntly ev~porated to dryne~. The crude peptide was puriied by gel chromatography (SEPHAD2X~ LH 20). 127 mg of pure product w~re obtained.

EXANPL~ 84 H-A~p-Ala-Val-~er-Tyr Gln-~y~-OH

- 24 - O.Z. 0050/40386 5.7 g of Fmoc-Lys(Boc)-~errifield resin (substitution O.35 mmol/g), corresponding to a batch ~ize of 2 mmol, were reacted as in AIb with 8 mmol each of Fmoc-Gln-OH Fmoc-Val-OH
E moc -Tyr ( tBu ) -OH Ehloc -Ala-OH
Fmoc-Ser-(Bzl)-OH Fmoc-A~p(OtBu)-OH

Subsequently, the t-butyl and Boc protective group~ were cleav~d off (steps 1-6 a3 in AIa). Th~ cyclization on the resin took place in NMP with the addition of 3.54 g of BOP and 3.5 ml of diisopropylethylamine (16 h). The peptide-re~in underwent N-terminal deprotection (steps 2-4 as in AIb) and drying under reduced pressure. The yield wa~ 6.4 g.

The crude product obtained after HF cleavage a~ in AII
wa~ purified by gel filtration (SEPHADeX G-15) and medium pre~ure chromatography (cf. ~IV; 10-30 % A;
0.25 % min~l). 23 mg of pure product were obtained.

The following can be prapared in a ~imilar manner to Example~ 83 and 84:

~ I
85~ Ac-Asp-Val-Ser-Tyr-Ly~-NK2 r ~
86 . Ac-Lys-VAl-S~r-Tyr-A8p-NH2 ~, 87. Ac-Ly~-Val-Ser-~yr-Asp-OH

88. Ac-Glu-Val-Ser-Tyr-Ly~-NH2 ~ I
89. A~-Glu-Val-SQr-Tyr-Ly~-OH
, - ~
90. H-Asp-VAl-Ser-Tyr-Ly~-OH
r 91. Ac-Lys-Val-Ser-Tyr-Glu-NH2 21~05060 - 25 - O~Z. 0050/40386 i g2. Ac-&lu-Val-Ser-Tyr-Orn-NH2 93. Ac-Asp-Val-Ser-Tyr-Orn-NH2 94. Ac-A3p-Gly-Ser-Tyr-Orn-NH2 _ j 95. Ac-r~ap-val-sex-Tyr-Asp-NH2 I
96. Ac-Asp-Val-Ser-Tyr-Dap-NH2 I
97. Ac-Dap-Val-Ser-Tyr-Glu-NH2 98. Ac-Glu-Val-Ser-Tyr-Dap-NH2 99. Ac-A~p-Ala-Val-Ser-Tyr-Gln-Ly8-NH2 100. Ac-A~p-Ala-Val-S~r Tyr-Gln-Lys-OH

101. Ac-Lys-Gly-Val-Ser-Tyr-Gln-A~p-NH2 102. Ac-~ys-Gly-~al-S~r-Tyr-Gln-A~p-OH

103. AC_A8P_A1a_Va1_Ser_~Yr_G1n_Orn-NH2 , I
104. Ac-Orn-Ala-Val-Ser-Tyr-Gln-A~p-N~2 r- ~ I
105. Ac-Arg-Ile-AI3p-Val-S~r-Tyr-$ys-Thr-Ly~-NH2 I
106. H-Ly~-Val-Gln-~yr-Asp-OH

107. Ac-Ly~-Val-Gln-Tyr-A~p-NH2 r -- l 108. Ac-Ile-Ly~-Val-Ser-Tyr-Glu-Thr-NH2 109. Ac-lle-Ly~-Val-Ser-Tyr-Glu-Thr-O~
~ ~
110. Ac-Asp-~le-Ser-Tyr-Orn-NH2 - 26 - O.Z. 0050/40386 111. Ac-Ile~-Arg-Asp-Ala-Val~-Tyr-Gln-Lys-Lys-Val-Axn-N~

112. Ac-Orn-Ala-D-Val-Ser-Tyr-Gln Asp-NH2 ~A1~-Val-Ser- ~ -Gln-Ab~1 0~5 g of Boc-Ab~-Merrifield resin (substitution mmol/g), corre~ponding to a batch ~ize of 0.5 mmol, wa~
reacted as in AIa with 2 mmol each of Boc-Gln-OH Boc-Val-OH
~oc-Tyr(~r-Z)-OH ~oc-Ala-OH
Boc-Ser(Bzl)-OH

After the synthe~is wa~ complete, ths peptide-re~in underwent N-ter~inal deprotection ~tsps 1-3 as in A~a) and sub~equent drying under reduced pres~ure. The yield wa~ 0.91 g.

~he crude product (290 mg) obtained after HF cleavage a~
in AII wa8 dis801ved in 500 ml of dega8~ed DMF. 210 mg of NaHCO3 and 660 mg of ~OP were added and then the mixture wa~ ~tirred at room temperature for 3 days. It wa~ then evaporated to drynes~, and the crude peptide was purified by gel chromatography (SEPHADEX~ LH 20). 138 mg of pure product were obtained.

Tyr-Gln-Thr-Ly~-Arg-Ile-Ala-V~l-Ser-r 1.11 g of Fmoc-S~r~t-Bu3-p-al~oxyb~nzyl alcohol-resin (sub~titution 0.45lamol/g), corre~ponding to a batch sizs of 0.5 ~mol, wexe reacted aa in A$b with 2 m~ol each of Fmoc-Val-OH Fmoc-Lys(Z)-OH

Z1~0~060 - 27 - O.Z. 0050/40386 Fmoc-Ala-OH Fmoc-Thr(tBu)-OH
Fmoc-Ile-OH Fmoc-Gln-OH
Fmoc-Arg(Tos)-oH Fmoc-Tyr (tBu)-OH

After the ~ynthesi~ wa~ complete, the peptide-re~in underwent N-terminal deprotection (steps 2-4 a in AIb) and cub~equent drying under reduced pre3sure. Ths yield was 1.8 g.

The crude peptide obtained after TFA cleavage as in AIII
wa~ dissolved ~n 500 ml of degassed DMF. 210 mg of NaHCO3 and 660 mg of BOP were added and the mixture wa~ then stirred at room temperature for 3 days. It wa~ then evaporated to drynQss, and the crude peptide was purified by gel chromatography (SEPHADEX~ LH 20). The isolated monomer (219 mg) wa~ deprotected as in A II and purified by medium pressure chromatography (cf. AIV; 40-60 ~ A;
0.25 % min~~). 68 mg of pure product were obtained.

The following can be prepared in a similar manner to Example~ 113 and 114:

115. rIle-Ala-Val-Ser-Tyr-Gln-T
116. rArg-Ile-Gly-Val-Ser-Tyr-Gln-Thr-~y 117. ~Ser-Ils-Ala-Val-Ser-Tyr-Gln-Thr-Ly~-Val 118. a-Val-Ser-Tyr-Gln-Gly 119. ~Ala-Val-Ser-Tyr-Gln-Bal 120. rAla-val-ser-Tyr-Gln-D-Aln ~ a-Val-Ser-~yr-Gln-Thr-Lys-D-Ala-Arg-Ile 121. ~le-Ala-Val-Ser-Tyr-Gln-Thr-D-Pr~

~Ala-Val-Ser-Tyr-Gln-3-Pr~

- 28 - O. Z . 0050/40386 122. ~Gly_Val_Ser_Tyr_Gln_Abs 123. rAla-Val-Ser-D-q~rr-Gln 124 . ~Gly-Val-Ser-q~yr-Gln-Thr 125. ~rg-Ile-Ser-Val-Ser-Tyr-Gln-Thr-Lys 10 126. rIle-Ala-Val-D-Ser-q!yr-Gln-127. rIle-Ala-Val-Ser-Tyr-Gln-Thr-D-Ala

Claims (8)

1. A peptide of the formula I, X-A-B-Tyr-Y I, where A is Ser, Val, Ile or Pro, B is a naturally occurring .alpha.-amino acid, X is G-NH-CHM--CO-, G-NH-CHN-CO-W-, G-R-NH-CHX-CO- or -G-R-NH-CHM-CO-W- and Y is -Z, -NH-CHQ-CO-Z, -V-NH-CHQ-CO-Z, -NH-CHQ-CO-U-Z
or -V-NH-CHQ-CO-U-Z
where, in X and Y, G is hydrogen or an amino-protective group, Z is OH or NH2 or a carboxyl-protective group or G and Z together are also a covalent bond or -CO-(CH2).-NH- where a i8 from 1 to 12, R, U, V and W are peptide chains composed of 1-4 naturally occurring .alpha.-amino acids, and M and Q are hydrogens or one of the following -CH(CH3)2, -CH(CH3)-C2H5, -CBH5, -CH(OH)-CH3, , or -(CH2)b-T

(with b being from 1 to 6 and T being hydrogen or OH, CH3O , CH3S, (CH3)2CH, C6H5 p-HO-C?H4, HS, H2N, HO-CO, H2N-CO or H2N-C(=NH)-NH) or M and Q together are a -(CH2)o-S-S-(CH2)d-, -(CH2)?-CO-NH-(CH2)f- or -(CH2)?-NH-CO-(CH2)?-NH-CO-(CH2)f- bridge (with c and d being from 1 to 4, e and f being from 1 to 6 and g being from 1 to 12), as well as the salts thereof with physiologically tolar-ated acids.

2. A peptide as claimed in claim 1, where G is hydrogen or an amino-protective group and Z is hydroxyl or amino or a carboxyl-protsctive group, and M and Q are not connected together.

3. A peptide as claimed in claim 1, where G is hydrogen or an amino-protective group and Z is hydroxyl or amino or a carboxyl-protective group, and M and Q together are a -(CH2)c-S-S-(CH2)d- bridge.

4. A peptide as claimed in claim 1, where G is hydrogen or an amino-protective group and Z is hydroxyl or amino or a carboxyl-protective group, and M and Q together are -(CH2)?-NH-CO-(CH2)f- or -(CH2)?-NH-CO-(CH2)g-NH-CO-(CH2)f.

5. A peptide as claimed in claim 1, where G + Z to-gether are a covalent bond or -CO-(CH2)?-NH-.

6. A peptide as claimed in claim 1 to 5 for use for controlling diseases.

7. The use of a peptide as claimed in claims 1 to 5 for controlling neoplastic diseases and autoimmune diseases as well as for controlling and preventing infections, inflammations and transplant rejection reactions.

8. A process for the preparation of a peptide as claimed in claims 1 to 5, which comprises preparation thereof using conventional methods of peptide chemistry.