CA2005056A1 - Tnf peptides - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE: Peptides of the formula X-Ala-His-A-Y, where A, X and Y are defined in the description, and the preparation thereof are described. The novel peptides are suitable for controlling diseases.

Description

20050~6 O. Z . 0050/40390 NOVEL TNF PEPTIDES

The present invention relates to novel peptide~ derived from tumor necro~is factor tTNF), the preparation thereof and the use thereof as dru~s.

Carswell et al. (Proc. Natl. Acad. Sci. USA 72 (1975~
3666) reported that the serum of endotoxin-treated animals which had previously been infected with the Calmette-Guerin ~train of Mycobacteria (BCG) brought about hemorrhagic necrosi~ in variou~ mouse tumors. ~his activity was ascribed to tumor necrosis factor. TNF also has a cytostatic or cytotoxic effect on a large number of transformed cell line~ in vitro, whereas normal human and animal cell lines are unaffected (Lymphokine Report~ Vol.

2, pp 235-275, Academic Press, New York, 1981). Recently, the biochemical characterization and the g~ne for human TNF have been described (Nature 312 (1984) 724, J. Biol.
Chem. 260 (1985) 2345, Nucl. Acids Re~. 13 (1935) 6361).

It i~ possible to deduce from thi~ data the following protein structure for mature human TNFs V~l~n~eD~r~ te~noD ~u~ysProVal~ sValVaLU3AY~Ro G~ uGl V~lGl. ~ ~ ~ D~IYalV~lP ~ Ll~lyl ~ l ~yrS
GlnV~l~Eh~nklyG~L~yCy~B~6~rrlio~V~lT~T~rUi~Ile S ~ gIl~l~lValSe~ n ~ ysV ~ lalleLy~SerPro CysG~dh~Glurhrbxf~uGlyAk~uAla~y~hl~kp~yCluProIld~deu GlyGly ValPhPr~l nT ~1 uLysGayAnpknyels L~f1UI1e~59Pn~O
~ ValTyrPh0GlyIleIleA~u The TNF gen~ of cattle, rabbits and mice have also been described (Cold Spring Harbor Symp. Quant. Biol. 51 (1986) 597).

BeYide~ its cytotoxic properties, ~NF i~ one of the main ~OIU50~
- 2 - o.Z. 0~g~J40390 sub~tances involved in inflammatory reaction~ (Pharmac.
Re~. 5 ~1988~ 1293. Animal models have shown that TNF is involved in septic shock (Science 229 ~1985) 869) and graft-vers~-ho~t disea~e (J. ~xp. Med. 166 (1987) 1280).

We have now found that peptides with a considerably lower molecular weight ha~e beneficial properties.

The present invention relàteq to peptide~ of the formula I
X-Al~-Hi~-A-Y
where A is Val, Leu, Ile or -NH-(CH2)m-CO- (with m being an integzr from 1 to 12), X is G-NH-CHM-CO-, G-NH-CHN-CO-W-, G-R-NH-CHM-CO- or G-R-~H-CH~-CO-W- and 5 Y is -3, -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 ~H2 or a carboxyl-prot~ctive group, or R is -Leu-Arg-Ser-Ser-Ser-Gln-Asn-Ser-Ser-Asp-Ly~-Pro-, -Val-Arg-Ser-Ser-Ser-Arg-Thr-Pro-Ser-Asp-~y~-Pro-, -Leu-Arg-Ser-Ser-Ser-Gln-Ala-Ser-Ser-Asn-Lys-Pro-, -Leu-Arg-Ser-Ala-Ser-Arg-Ala-LQu-Ser-Asp-Lys-Pro-or a ~equence of 5-11 amino acid re~idue4 from ona o~ the~e peptide chains or a-chain composed of 1-4 naturally occurring ~-amino acids, U, V, and W are chain~ compos~d of 1-4 naturally occur-ring ~-amino acid~, and M and Q are hydrogens or on~ of the following -CH(CH3)2, -CH(CH3)-C2H5, -C6H5, -CH(OH)-CH3 ~H 2~ ~H Z~ or --t CH 2 ) b--T
H

(with b b0ing from 1 to 6 and ~ ~eing hydrogen or '~C~5056 - 3 - O. Z . 0050/40390 OH, CH30, CH3S ~ ( CH3 ) zCH ~ C6X5 ~ p-HO--CbH4, HS, H2~, HO-CO, H2N-CO or H2N-C ( =N~ ) -NH ) or M and Q together are a - ( CH2 ) c-S-S- ( CH2 ) d- ~ - ( CH2 ) ,-CO-~3H-( CH2 ) ~- or - ( CH2 ) "-NH-CO- ( CH2 ) ~-NH-CO- ( CH2 ) ~- 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), a~ well a~ the ~alt~ thereof with physiologically tolerated acids.

~he peptide of the formuls I are constructed of ~-amino acid~, but they can contain 1 or 2 D-a~ino acids. The side-chains of the trifunctional amino acids can carry protective group3 or be unprotected.

Particularly preferred phy~iologically tolerated acid~
ares hydrochloric acid, citric acid, tartaric acid, lactic acid, phosphoric acid, methanesulfonic acid, acetic acid, formic acid, male~c acid, fumaric acid, malic acid, succinic acid, malonic acid, ~ulfuric acid, L-glutamic acid, L-aspartic àcid, pyruvic acid, mucic acid, benzoic acid, glucuronic ac~d, oxalic acid, ascor-bic acid and acetylglycine.

The novel peptide~ can be open-chain (G 3 H, 2mino-protective group; Z = OH, NH2, car~oxyl-protective sroup M and Q not connected together) and, in particular, ha~e a disulfide bridgQ (G z H, amino-protective group;
z D 0~ NH2~ carboxyl-protective group; ~ + Q = -(CH2)C-S-S-(CH2)d-) or a side chain bridge (G = H, amino-protec-tive group, Z - OH, NH2, car~oxyl-protectiYe group, ~ ~ Q
= -(CH2),-NH-CO-(CH2)s~ or -(CH2).-NH-CO-(CH2)~-N~-CO-( CH2 ) ~

The novel compound~ can be prepared by conventional methods of peptide chemi~try.

~hu~, the pept$des can be con~tructed sequentially from _ 4 _ o z. 0050/40390 amino acids or by linking together suitable smaller peptide fragments. In the sequential construction, the peptide chain is extended stepwi~e, by one amino acid each time, starting at the C terminus. In the case of coupling of fragments it is possible to link together fragments of different length~, these in turn being obtainable by sequential construction from amino acids or coupling of other fragments. The cyclic peptides are obtained, after ~ynthesi~ of the open-chain peptide~, by a cycliza~ion reaction carried out in high dilution.

In the ca~e both of sequential construction and of fragment coupling it i~ nece~sary for the building block~
to be linked by formation of an amide linkage.
Enzymatic and chemical method3 are suitable for thi~.

Chemical method~ for forming amide linkages are dealt with in detail by M~ller, Methoden der Organi~chen Chemie (Method~ of Organic Chemistry) Vol. XV/2, pp 1-364, Thieme Verlag, Stuttgart, 1974; Stewart, Young, Solid Phase Peptide Synthe~i~, pp 31-34, 71-82, Pierce Chemical Company, ~ockford, 1984; Bodan~zky, ~lausner, Ondetti, Peptide Synthesis, pp 85-128, John Wiley & Sons, New York, 1976 and other ~tandard works of peptide chemistry.
Particularly preferred are the azide method, the ~ymmetr-ical and mixed anhydride method, active e8t8r8 generated in situ or preformed and the formation of amide linkageA
u8ing coupling reagent~ (activator~), in particular dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), l-~thyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), n-propanephosphonic anhydride (PPA), N,N-bi~(2-oxo-3-oxazolidinyl)amidopho~phoryl chloride (BOP-Cl), diphenylpho~phosyl azide (DPPA), Castro's reagent (BOP), O-benzotriazolyl-~,N,N~,N~-tetra-methyluron~um 8~1t8 (HBTU), 2,5-diphenyl-2,3-dihydro-3-3~ oxo-4-hydroxythiophone dioxide (Steglich'3 reagent;

_ 5 _ o.z. 0050/40390 HOTDO) and l,l~-carbonyldiLmidazole (CDI). The coupling reagents can be employed alone or in combination with additives such as N,N'-dLmethyl-4-aminopyridine (DMAP), N-hydroxybenzotriazole (HOBt), N-hydroxybenzotriazine (HOOBt), N-hydroxysuccinLmide (HOSu) or 2-hydroxy-pyridine.

Where~s it i~ normally po~sible to di~pense with protec-tive group~ in enzymatic peptide ~ynthesis, for chemical synthe~i~ it is neceqsary for there to be reversible protection of the reactive functional groups which are not involvèd in the formation of the amide linkage on the two reactant~. Three conventional protective group technique~ are preferred for chemical peptide synthe~es:
the benzyloxycarbonyl (Z), the t-butyloxycarbonyl (Boc) and the 9-fluorenylmethyloxycarbonyl (~moc~ techniques.
In each ca~e the protective group on the -amino group of the chain-extending building block is identified. The ~ide-chain protective groups on the trifunctional amino acids are cho~en 80 that they are not necessarily elimin-ated together with the ~-amino protective group. A
detailed review of amino acid protective group~ i~ gi~en by ~ller, Nethoden der Organischen Che~ie Vol XV/l, pp 20-906, Thiem~ Verlag, Stuttgart, 1974.

The building blocks used to construct the peptide chain can be reacted in solution, in suspension or by a method simllar to that de~cribed by Merrifield in J.Amer. Chem.
Soc. 8~ (1963) 2149. Particularly preferred methods are those in which peptide~ are constructed ~equentially or by fragment coupling by use of the Z, Boc or Fmoc protec-tive group technique, in which case the reaction takesplace in ~olution, a~ well as tho~e in which, similar to the Nerrifield technique, one reactant i9 bound to an in~oluble polymeric Rupport ~al~o called re~in herein-after). ~hi~ typically entails thQ peptide being con-~tructed ~equentially on the polymeric ~upport, by use of 0~5 ~
- 6 - O.Z. 0050/40390 the Boc or Fmoc protective group technique, with the growing peptide chain being covalently bonded at the C terminus to the insoluble resin particle~ (cf. Figures 1 and 2). This procedure allow~ reagent~ and byproducts to be removed by filtration, and thus recrystallizatio~
of intermediates is superfluou~.

The protected amino acids can bs bonded to ~ny ~uitable polymers which merely need to be in~oluble in the ol-vent~ used and to have a table physical form which allows easy filtration. The polymer mu~t contain a functional group to which the fir~t protected amino acid can be firmly linked by a covalent bond. A wide variety of polymers i8 suitable for thi~ purpose, for example cellulose, polyvinyl alcohol, polymethacrylate, sulfon-ated polystyrene, chloromethylated copolymer of ~tyreneand divinylbenzen~ lMerrifield resin), 4-methylbenz-hydrylamine-resin (MBHA-resin), phenylac~tamidomathyl-re~in (Pam-resin), p-benzyloxybenzyl alcohol-resin, benzhydrylamine-re~in (BHA-resin), 4-hydroxymethyl-benzoyloxymethyl-re~in, the resin u3ed by Breipohl et al.
(Te~rah~dron Lett. 28 (1987) 565; from ~ACHEM), HYCR~M
re~in (from ORPEG~N) or SASRIN resin (from BACHEM~.

Solv6nts suitable for peptide synth2~is in solution are all those which are inert under the resction condition~, in particular water, N,N-di~ethylformamide (DMF), dimethyl sulfoxida (DMS0), acetonitrile, dichlorome~hane (DCM), 1,4-dioxane, tetrahydrofuran (THF), N-methyl-2-pyrrolidone (N~P) and mixturea of the said solvents.
Peptide synthesis on polymeric suppor~ can be carried out in all inert organic solvents which dis~olve ~he amino acid derivatives u~ed; however, solvents which have re3in-~welling propsrtie~ are preferred, ~uch a~ DMF, DCM, NMP, acetonitrLlo and DMS0, as well a~ mixtures of the~o solvent~.

;~005056 - 7 - O.Z. 00~0/40390 After the peptide has been synthesized it is cleaved off the polymeric support. The cleavage condition~ for the various type~ of resins are di~closed in the literature.
The cleavage reactions most commonly use acid and pa71adium catalysis, in particular cleavage in anhydrous liquid hydrogen fluoride, in anhydrous trifluoromethane-sulfonic acid, in dilute or concentrated trifluoroacetic acid or palladium-catalyzed cleavage in THF or T~F-DCM
mixtures in the presence of a weak ba~e such as morpho-line. The protective groups may, depending on the choicethereof, be retained or likewi~e cleaved off under the cleavage conditions. Partial deprotection of the peptide may also be worthwhile if the intention i8 to carry out certain derivatization reaction~ or a cyclization.

Some of the novel peptide~ have good cytotoxic proper-tie~. Some others of the peptide~ ha~e high affinity for the cellular TNF receptor without, however, having cytotoxic activity. ~hey are therefore TNF antagonist~.
They compete with natural TNF for binding to the cellular TNF rereptor and thus ~uppres~ the TNF effect. The novel peptide~ are valuable drugs which can be employed for treating neopla tic di~ease~ and autoi~mune diseases a~
well a~ for controlling and preventing infection~
inflammations and transplant re~ection reactions. Simple experiment~ can be used to elucidate the mode of action of ths indi~idual peptide~. Th~ cytotoxicity of the peptid~ is determined by incubating a TNF-~ensitive cell line in the presence of the peptide. In a ~econd experi-mental approach, the cell line i8 incubated with the relevant peptid~ in the presence of a lethal amount of ~NF. It is possible in this way to detect the $NF-antagon~tic effect. In addition, the affinity of the peptid~ for the cellular TNF receptor i~ deter~ined in an in vitro binding experiment.

The follo~ing te~t ~y~tems ware used to ch~r~cterize the 200505~
- 8 - O.Z. 0050/40390 agonistic and antagonistic effects of the novel peptide~:

I. Cytotoxicity test on TNF-sensitive indicator cells, II. Cytotoxicity antagonism te~t on TNF-~ensitive indicator cell~, III. Competitive receptor-binding te~t on indicator cell~
expressing TNF receptor.

I. Cytotoxicity te~t The agoni~tic effect~ of the novel peptides are a~sessed on the ba3i~ of their cytotoxic effect on TNF-~ensitive cells (e.g. L929, MCF-7, A204, U937).
The te3t with L929 and NCF-7 wa~ carried out a~
followss 1. 100 ~1 of culture medium containing 3 to 5 x 103 fra~hly trypsiniz~d, exponentially growing, L929 cell~ (mou~e) or MCF-7 cells (human) were pipetted into the wells of a 96-well flat-bottom culture plate. The plate was incubated at 37C
overnight. ThQ air in the incubator was saturated with water vapor and contained 5% CO2 by volume.

The L929 culture medium contained 500 ml of lx ~arle's MEM (Boehringer ~annheim), 50 ml of heat-nactivated (5SC, 30 min) fetal calf 8erUm (FCS), 50 ml of L-gluta~ins (200 mM), 5 ml of lOOx non-e~sential amino acid~, 3 ml of LN HEPES
buffer pH 7.2,and 50 ml of gentamicin (50 mg/ml).

The MCF-7 culture medium contained 500 ml of lx Dulbecco's MBM (~oehringer Mannheim), 100 ml of heat-inactivated t56-C, 30 min) FCS, 5 ~1 of ~-glutamine and 5 ml of lOOx non-e3sential amino acids.

2. Th~ next day 100 ~1 of the peptide ~olution to be ;~05056 _ g _ o.z. 0050/40390 tested were added to the cell cultures and sub~ected to serial 2-fold dilution. In addition, some cell control~ ti.e. cell culture3 not treated with peptide dilution) and some rhu-~NF
controls (i.e. cell cultures treated with recom-binant human ~NF~ were also made up. The culture plate wa~ incubated at 37~C in an atmosphere of air saturated with water vapor and containing 5%
C02 by volume for 48 h.

3. The percentage of ~urviving cell~ in the cultures treated with peptLde dilution was determined by staining with cry~tal viole~. For this purpose, the liquid wa~ removed from the well~ of the test plate by tapping it. 50 ~1 of crystal violet solution were pipetted into each well.

The composition of the crystal violet ~olution wa~ as follows:

3.75 g of cry~tal violet 1.75 g of NaCl 161.5 ml of ethanol 43.2 ml of 37% formaldehyde water ad 500 ml The crystal violet solution wa~ left in the wells for 20 min and then likewi~e removed by tapping.
The plates were then washed 5 times by immersion in wat~r in order to remove dy~ not bound to the cells. Tho dye bound to tha cells was extracted by adding 100 ~1 of reagent solution (SOS etha-nol, 0.1~ glacial acetic acid, 49.9~ water) to each well.

4. The plata~ were ~haken for 5 min to obtain a ~olution of uniform color in each well. The 5 ~ S 6 - 10 - O.Z. 0050/40390 ~urviving cells ware determined by mea~uring the extinction at 540 nm of the colored solution in the individual wells.

5. 5ubsequently, by relating to the cell control, the 50% cytotoxicity value was definet, and the reciprocal of the sample dilution which resulted in 50~ cytotoxicity was calculated a~ the cyto-toxic activity of the te~t sa~ple.

II. Cytotoxicity antagonism test The antagonistic effect of the peptide~ wa3 a~ses~ed on the basis of their property of antagonizing the cytotoxic effect of rhu-TNF on TNF ~en~itive cell~
(e.g. L929, ~C~-7, A204, U937). The cytotoxicity antagonism te~t with L9~9 and MCF-7 cells wa3 carried out as follow~s 1. 100 ~1 of culture medium containing 3 to 5 x 103 fre~hly tryp~inized, exponsn~ially growing, Lg29 cells (mouse! or MCF-7 cell~ (human) were pipetted into the wells of a 96-well flat-bottom culture plate. The plate wa~ incubated at 37C
overnight. The air in the incubator wa~ ~aturated with water vapor and contained 5% CO2 by volume.

The L929 culture medium contained 500 ml of lx Earle's MEM (Boehringer Ma~nheim), 50 ml of heat-inactivated (56-C, 30 min) FCS, 5 ml of ~-gluta-min~ (200 mM), S ml of lOOx non-essential amino acid~, 3 ml of lM ~EP8S ~uffer pN 7.2, and 500 ~1 of ~entami~in (50 m~/ml).

ThQ N~F-7 culture mediu~ contained 500 ml of lx DulbQcco's MEM (Boehringer Nannheim), 100 ml of heat-i~activated (56-C, 30 min) FCS, 5 ml of L-glutamine (200 mM) and 5 ml of lOOx non-~sen-tial amino acid~.

~305056 ~ O.Z. 0050/40390 2. The next day 10~ ~1 of the peptide ~olution to be te~ted were added to the cell culture~ and sub~ected to ~erial 2-fold dilution. Then, 100 ~1 of a rhu-TNF dilution in culture medium, which dilution had an 80-100% cytotoxic effect in the . final concentration in the cell culture, were added to these cell cultures. In additlon, ~ome cell controls (i.e. cell cultures not treated with peptide solution or with rhu-TNF solution) and ~ome rhu-TNF controls (= cell cultures treated only with rhu-TNF solution) were also made up. The culture plate wa3 then incubated at 37~C in an atmosphere of air ~aturated with water vapor and containing 5% CO2 by volume for 48 h.

3. ~he percenta~e of surviving cell3 in the cultures treated with substance solution wa~ determined by ~taining with cry~tal violet. For this purpose, the liquid wa~ removed from the wells of the test plate by tapping Lt. 50 ~1 of cry~tal violet ~olution were pipetted into each well.

The crystal violet ~olution had the composition specif~ed in I.3 The crystal violet solution was left in the wells for 20 min and then likewisQ removed by tapping.
The plates were then washed 5 time~ by immer~ion in water in order to remove dye not bound to the cell~. ~he dye bound to the cells wa~ extracted by adding 100 ~1 of reagent ~olution (S0~ etha-nol, 0.1~ glaciAl acetic acid, 49.9% water) to each well.

4. The plate~ were shaken for 5 min to obtain a solution of uniform color in each well. The ZIQ0~0~6 - 12 - O.Z. 0050/40390 surviving cells were determined by mea~uring the extinction at 540 nm of the colored solution in the individual well~.

5. Subsequently, by relating to the cell control and the rhu-TNF control, the 50% antagoni~m value wa~
defined, and the ~ample concentration which resulted in 50% antagonism of rhu-TNF cytotox-icity at the rhu-TWF concentration u~ed was calculated a~ antagonistic activity of the ~ample te~ted.

III. Competitive receptor-binding te~t Both the agoni~tic and antagoni~tic effect~ of peptide~ are conditional on th~ latter binding to the TMF receptor. Thi~ mean~ that peptides with an agonistic or antagoni~tic effect compete with rhu-TNF for binding to the TNF receptor on TNF-sensitive indicator cells (e.g. U937). ~he competi-tive receptor-binding te~t wa~ carriad out a~
follow~ 5 1. 100 ~l of medium containing various concentra-tions of the peptide to b~ te~ted and of rhu-T~F
(= control) were pipetted into the reaction vessel~. The medium comprised 500 ~1 of PBS
(Boehringer Mannheim), lO ml of heat-inactivatsd (56C, 30 min) FCS and 100 mg of sodium azide.

2. Sub~equently, lO0 ~l of medium containing 1 ng of ~Z~I-labeled rhu-~NF (Bol~on lactoperoxida~e method) were placed in the reaction ve~8els and mixed. The non-specific bindinq (N5B) was deter-~ined by ~ixing in the reaction ve~8el~ the l2~I-labeled rhu-TNF (1 ng of l2~I-rhu-TNF in lO0 ~l of medium) with a 200-fold exce~8 of unl~beled rh~-TNP (200 ng of rhu-TWF in 100 ~l of medium).

21~(~50S~

- 13 - O.Z. 0050/40390 3. Then 100 ~1 of medium containing 2 x 10~ U937 cell~ (human) were pipetted into the reaction ve~sel~ and mixed. The reaction vessel3 (test volume 300 ~1) w~re incubated at 0C for 90 min.
The reaction mixtures were remixed after 45 min.

4. After the incubation the cells were centrifuged at 1800 rp~ and 4C for 5 min, washed 3 times with medium and transferred quantitatively into counting vial~, and the cell-bound radioactivity wa~ determined in a Clini gamma counter 1272 (LXB Wallac).

5. After the mea~uremant~ had ~ee~ corrected for the non-specific binding, thè 50% competition value w~ defined by relat~on to th~ overall binding, and tha ~ample concentration which led to 50%
competition of l25I-rhu-TNF binding at the125I-rhu-TNF concentratio~ used wa~ calculated as the competitive activity of the ~ample tested.

The Example~ which follow are intended to ~xplain the invention in more detail. The proteinogenous a~ino acids are abbreviated in th~ ~xample8 using the con~entional three-l~tter code. Other meaning~ ares Ab~ = 4-aminobutyric acid, ~c - acetic acid, Ade - 10-amlnodecanoic acid, Ahx = 6-aminohexanoic acid, A~o ~ g_~minononanoic acid, Aoc = 8-aminooctanoic acid, Ape - 5-aminopentanoic acid, ~cy - homocyYteine, Hly ~ homolysine, Orn - ornith~ne, Dap - 2,3-diaminopropionic acid.

A. General procedure I. The peptides claim2d in clai~ 1 were ~ynthesized using ~tandard mothod~- of solid-pha~e p~ptide ynthe~is in a compl~tely au~o~tic mod~l 430A

Z~3~505~
- 14 - O.Z. 0050/4039~
synthe~i~ in a completely automatic model 430A
peptide synthesizer from APPLIED BIOSYSTEMS. The apparatus uses different synthesis cycle~ for the Boc and Fmoc protective group techniques.

a) Synthesis cycle for the Boc protective group technique 1. 30% trifluonwLetic acid in DCM 1 x 3 mun 2. 50% triflu=nYY~oic acid in ~CM 1 x 17 mLn 3. DCMwashing 5 x 1 min 4. 5% diL~QylethyL~ne in DCM 1 x 1 min 5. 5% dii~rpylethyla~Ie in NMP 1 x 1 min 6. NMP was~ing 5 x 1 min 7. Addition of pn~tivated prJls~Id a~
acid (activation ky 1 eqlivalent of DOC
and 1 equivalent of HOBt in NMP/DCM);
pepkide coupling (lst part) 1 x 30 min 8. A~ition of nMSO to the n~ n mi~h~e u~l it c ~ 20% nMSO by volume 9. Pq~i~b ooup~ng ~2n~ ~ t) 1 x 16 min l0.Ad~iticn of 3.8 ~YZ~ of d~

11. p~ e coupling (3rd Eart) 1 x 7 min l2.DCMwad~ng 3 x ln~n 13.1f r3x~cn i~ ~xxoplete, rq~ition of coup~ (neturn bo 5.) 14. l0~ ~o~c a~ride, 5% dilx}D~Qyl-ethyL~ne in DCM 1 x 2 min 15. 10% ao~i~ a~ride Ln DC~ 1 x 4 min 16.DoMwa~n~ 4 x l min 17 R~lIn to l.

b) Sy~is cy~le for ths Fmoc p~æ~ive ~x~ toc~l4Fe 1. N~P w~*~ng 1 x l min 2. 20% p~ ne in NMP 1 x 4 min 3. 20% plp3i~lne in NMP 1 x 16 m~n 4. NMP wa~hing 5 x 1 min ~;~G0505;~
- 15 - O.Z. 0050/40390 5. A~dition of pn~tivabed pr~lr~ed acid (a~tivation by 1 eqyivalent of DOC
and 1 ~ alent of HOBt in NMP/DCM);
p~lde coupling 1 x 61 min 6. NMP wzshin~ 3 x 1 min 7. ~f r3~0n i ~Yx~plete, ~ition of coupling (~ n to 5.) 8. 10% ~ a~ride in NMP 1 ~ 8 min 9. NMP w2shing 3 x 1 min 10. R~n bo 2.

II. Working up of peptide-re~ins obtained a~ in Ia The peptide-resin obtained a~ in Ia was drisd under reduced pressure and tran~ferred into a reaction ves~el of a Teflon HF apparatus (from P~NINSULA).
Addition of a scavenger, preferably ani~ole (1 ml/g of re~in), and of a thiol, in the ca~e of tryptophan-containing peptide~, to remove the indole formyl group, preferably ethanedithiol (0.5 ml/g of re~in)~
was followed by condensation i~ of hydrogen fluoride (10 ml/~ o resin) while cooling with liquid N2. The mixture waa allowed to wPrm to O-C, and waQ ~tirred at thi~ temperature for 45 min. The hydrogen fluor-id~ wa~ then stripped off under reduced pre~aure a~d the residue wa~ wa~hed with e~hyl acetate in order to rem~ve remaining 3cavenger. The peptid~ w~
extracted with 30% stren~th acetic acid and filtered, and the filtrate was freezQ-dried.

To prepare peptide hydr~zides, the peptlde-resin (Pam- or Merrifield resin) was ~u~pended in DMF
(15 ml/g of re~in), hydrazine hydrate (20 equiva-lenta) was added, and the mixtur~ w~s stirred at room t~mpsra~ure for 2 day~. To work up, the reain was filtered off and the filtrate wa~ evaporated to dry~ess. The reaidue was cry~tallized from DM~/~t2O
or ~eOH/Et2O.

:, .

;?dO05056 - 16 - O.Z. 0050/40390 III. Working up of the peptide-resin~ obtained as in Ib ~he peptide-resin obtained as in Ib was dried under reduced pressure and subYequently sub~ected to one of the following cleavage procedures, depending on S the amino acid compo~ition (Wade, Tregear, ~oward Florey Fmoc-Workshop Manua1, ~el~ourne 1985).

2~3~SOS~j - 17 - O.Z- 0050/4039 Peptide containing Clea~age conditions Arg(Mtr) Met Trp TFA Scavenger Reaction Time S _ no no no 95% 5% H20 1.5 h yes no no 95% 5% thioanicole > 3 h no yes no 95~ 5~ ethyl methyl 1.5 h sulfide no no yes 95% 5% ethanedithiol~ 1.5 h anisole (1:3) no yes yes 95% 5% ethanedithiol/ 1.5 h ani~ole/ethyl methyl sulfide (1:3:11 ye~ yes ye~ 93% 7% ethanedithiol/2 3 h anisole/ethyl methyl ~ulfids (1:3:3) The ~uspension of the peptide-resin in the suitable TFA mixture was stirred at room temperature for the stated time and then the resin was filtered off and wa~h~d with TFA and with DC~. The filtrate and the wa~hings were exten~ively conce~trated, and the peptide was precipitated by addition of diethyl ether. The mixture wa~ cooled in an ice bath, and the precipitat~ was filtered off, taken up in 30%
acetic acid and freeze-dried.

IV. Purification and characterization of the peptides Purification wa~ by gel chromatography (SEPHAD~X~
G-10, G-15/10% HOAc; SEPHAD~X~ LH20/MeOH) and 8ub-sequent medium prs~sure chromato~raphy (~tationary pha~e: HD-SIL C-18, 20-45 ~, 100 ~; mobile phase:
gradient with A = 0.1% TFA/MeOH, B = 0.1% TFA/H20).

- 18 - O.Z. 0050/40390 The purity of the final products wa3 detelmined by analytical HPLC (~tationary pha~e: 100 x 2.1 mm VYDAC C-18, 5 ~, 300 ~; mobile phase = CH3CN/H20 gradient buffered with 0.1% TFA, 40C). Charac-S terization was by means of amino acid analy~i~ and fast atom bombardment ma3s spec~rometry.

B. Specific procedures Ac-Pn~br-Asp-~ys-kx~Val-Ala-Hi~-Ap~y-Ile-Ile-Ala-Leu-CH

1.11 g o~ Boc-~eu-Pam-resin ~ub~titution 0.45 mmol/g), corresponding to a batch size of 0.5 mmol, were reacted in as in AIa with 2 mmol each of Boc-Ala-OH Boc-Hi8(Z)-OH ~oc-Lys(Cl-Z)-OH
Boc-Ile-OH Boc-Ala-OH Boc-Asp(OChx)-OH
Boc-Ile-OH Boc-Val-OH Boc-Ser(Bzl)-OH
Boc-Gly-OH Boc-Pro-OH Boc-Pro-OH
Boc-Ape-OH

(~tep~ 14-16 were di~pensed with in all the couplings follo~ing His).

After th~ synthesi~ was co~pletec the N terminus was acetylated (8tep~ 1-6 ~nd 14-16 a~ in AIa) and the peptide-resin w~ dried under reduced pressure; the yield was 1.91 g.

0.95 g of the re~in obtained in this way was ~ub~ected to HF cleavage as in AII. The crude product (245 mg) was purified ~y gel filtration (SEPHAD~ G-10) and medium presQure chro~atography (cf. AIV; 60-80% A; 0.25~ min~l).
127 mg of final prod~ct were obtained.

'~C~ 5 U ~i - 19 - O.Z. 0050/40390 ~-Val-Arg-Ser-Ser-Ser-Arg-Thr-Pro-Ser-Asp-LYS-PrO-Val-Ala-Hi~-Aoc-Gly-Ile-Ile-Ala-Leu-OH

0.48 g of Fmoc-~eu-p-alkoxybenzyl alcohol-resin (sub~tit-S ution 0.52 mmol/g~, corresponding to a batch size of 0.2S mmol, was reacted a~ in AIb with 1 mmol each of Fmoc-Ala-OH Fmoc-Val-OH Fmoc-Arg(Mtr)-OH
Fmoc-Ile-OH Fmoc-Pro-OH Fmoc-Ser(tBu)-OH
Fmoc-Ile-OH Fmoc-Ly~(Boc)-OH Fmoc-Ser(tBu)-OH
Fmoc-Gly-OH Fmoc-A~p(OtBu)-OH Fmoc-Ser(t~u)-OH
Fmoc-Aoc-OH Fmoc-Ser(tBU)-OH Fmoc-Arg~Mtr)-OH
Fmoc-His~Trt)-OH Fmoc-Pro-OH Fmoc-~al-ON
Fmoc-Ala-OH Fmoc-Thr(tBU3-OH

After the synthesis wa~ complete, th0 N terminu~ wa~
deprotected (step~ 2-4 a~ in AI~). The resulting peptide-resin was dried under reduced pressure; thQ yield was 1.24 g.

The crude peptide (475 mg) obtain~d after TFA cleavage as in AIII wa~ purified by gel filtr~tion (SFPHADEX~ G-10) and medium pressur~ chromatography (cf. AIV; 50-70~ A;
0.25% min~';). 197 mg of pure product were obtained.

The following can be prepared in a 8~m~ lar manner to Examples 1 and 2:

3. H-Vla-A~br~r~r-Arg-lhr-P~br-AsF-Ly~-Pro-Val-Ala-Hi~-~p~y-Ile-Ile-Ala-Ieu-oH
4. Ac-Val-A ~ -Ser-S3r-An~hr-Pn~br-A~p-Ly~-Pro-Val-Ala-His-Ap~y-Ile-Ile-Ala-Lsu-N~
5. Ac-Pn~bo~Aop-Ly~-P~o-Val-Ala~ p~y-~Ile,Ala-leu-N~
6. Ac-Pn~br-A~p-Ly~-Pro-V~l-Ala-~R-Aoc~Gly-Ile-Ile-AL~-T~-oH
7. H~Ser-A~2-Ly~-Prc-V~l-Ala~ p~y-Ile_~Ala-Ieu-Cff S0S~;
- 20 - O.Z. 00~0/40390 8. Ac-SQr-A~p-Lys-~Val-Ala-Hi~-Ap~y-Ile-I~e-Ala-Leu-oH
9. Ac-Ser-Asp-Lys~x~Val-Ala-His-Ap~ly-Ile-Ile-Ala-Leu-~

EXA~LE 1 0 H-Asp-Lys-Pro-Cys-Ala-His-Ape-Gly-Cys Ile Ala-Leu-OH
1.11 g of Boc-Leu-Pam-resin (substitution 0.45 mmol/g), corresponding to a batch size of 0.5 mmol were reacted a~
in AIa with 2 mmol each of Boc-Ala-OH Boc-Ape-O~ Boc-Pro-O~
Boc-Ile-OH Boc-Hi8(Z)-ON Boc-Ly~(Cl-Z)-OH
Boc-Cy~(pMB)-OH Boc-Ala-OH Boc-A~p(OChx)-OH
Boc-Gly-OH Boc-Cys(p~B)-OH

(steps 14-16 were dispensed with in all the couplings following Hi3).

The re~ulting peptide-resin was dried under reduced pres~urQ; the yield wa~ 1.76 g.

0.88 g o the re~in obtained in this way wa~ sub~ected to HF cleavage a3 in AII. The ~reeze-dried crude product was tsken up in 2 1 of 0.1% strength ace~ic acid, and the pH
wa~ then ad~u~ted to 8.4 with aqueous ammonia. Under an argon atmo~phere, 0.01 N g3[Fe(CN)a~ ~olut~on wa~ ~lowly added dropwise until the yellowiah-green color parsi~ted for at l~a~t 15 min. Tha mix~ure was then stirred for ~ h ~nd then acidified to pK 4.5 with glacial ace~ic acid, and 15 ml of ~n aqueous suspension of ~n an$on exchanger (BIORAD- 3 x 4A, chloride form) were added. After 30 min, the ion exchAnger re~in wa~ filtered off, and the filt-rate wa~ concentrated to 100 ml in ~ rot~ry evaporator and ~ub~equently freeze-dried.

;~G~35l)5t;

- 21 - O.Z. 0050/40390 All the sol~ent~ u~ed had previously been ~aturated with nitrogen in order to prevent any oxidation of the free cyqteine residues.

The crude product wa~ purified by gel chromatography S (SEPHADEX~ G-15) and medium pressure chromatography (cf.
AIV; 60-80% A; 0.25~ minl). 72 mg of pure product were obtained.

The following can be prepared in a ~imilar manner to Example lO (p-~BHA-re~in wa~ used for the prepara~ion of the peptide amides):

Il. Ac-C~ls-Ala-HiS-Val-C~5-NH2 12. Ac-Cys^Ala-H1s-Leu-C~s-NHf 13. Ae-Cys-Ala-H1S-~t--CYs~NH2 14. Ac-C~s-Al~-H1s-Aoc-Cys-NH2 15. Ac-C~s-Al~-H1s-Ano-Cys-NH2 16. ~c-Cys-Al~-H1s-Ade-Cys-Nh2 17. H-C~S-AI~-H1S-A~S-Gly-Cys-OH

18. Ae-Cys-~ H1s-Abs-Gty-Cys-~H2 I9. ~-CyS-~la-~ls-~po-61y-CyS-OH
20. Ac-Cys-At~-H1s-~p--6ty-Cys-NH2 21. H-C~s-Al~-H1s-~hx-GI~-C~s-OH

22. Ac-Pro-C~s-Ala-Hls-Ap~-6l~-Cys-ll--N~2 " 2~05056 - 22 - 0~ Z . 0050/40390 23. Ac-Lys-Pro-Cys-Ala-His-Ape-Gly-Cys-lle-AI~-NH2 24. H-Asp-Lys-Pro-Cys-Ala-His-Abs-Gly-Cys-lle-Ala-Leu-OH
25. Ac-Asp-Lys-Pro-Cys-Ala-His-Abs-Gly-Cys-lle-Ala-Leu-O~
26. H-Asp-Lys-Pro-Cys-Ala-His-Abs-Gly-Cys-lle-Ala-Leu-NH2 27. Ac-Asp-Lys-Pro-Cys-Ala-His-Abs-Gly-Cys-Ile-~la-Leu-NH2 28. H-Asp-Lys-Pro-Hcy-Ala-His-Abs-Gly-Cys-lle-Ala-Leu-OH

29. Ac-Asp-Lys-Pro-Hcy-Ala-His-Abs-Gly-Cys-lle-Ala-Leu-NH2 30. H-Asp-Lys-Pro-Cys-Ala-His-Abs-Gly-Hcy-lle-Ala-Leu-OH

31. Ac-Asp-Lys-Pro-Cys-Ala-His-Abs-Gly-Hcy-lle-Ala-Leu-NH2 32. H-Asp-Lys-Pro-Hcy-Ala-His-Abs-Gly-Hcy-lle-Ala-~eu-OH
33. Ac-Asp-Lys-Pro-Hcy-Ala-H3s-Abs-Gly-Hcy-lle-Ala-Leu-NH2 34. Ac-Asp-Lys-Pro-Cys-Ala-His-Ape-Gly-Cys-~le-Ala-Leu-OH
, _ .
35. H-Asp-Lys-Pro-Cys-Ala-H3S-Ape-Gly-Cys-lle-Ala-Leu-NH2 36. Ac-Asp-Lys-Pro-Cys-Ala-tl3s-Apo-Gly-Cys-lle-Ala-Leu-NH2 37. H-Asp-Lys-Pro-Hcy-Ala-H3s-Ap~-Gly-CyS-lle-Ala-Leu-OH
i 38. Ac-Asp-Ly5-Pro-Hcy-Ala-His-Ape-Gly-Cys-llc-Ala-Leu-NH2 39. H-Asp-Lys-Pro-Cys-~la-His-Apa-Gly-Hcy-Ilo-Ala-Leu-OH
40. Ac-Asp-Lys-Pro-Cys-Ala-H~s-Ape-Gly-Hcy~ -Ala-Leu-NH2 r 41. H-Asj-Lys-Pro-Hcy-~la-H~s-Apo-Gly-Hcy-lle-Ala-Leu-OH
I_ , 42. Ac-Asp-Lys-Pro-Hcy-Ala-His-Ape-Gly-Hcy-lle-Ala-Leu-NH2 43. H-Asp-Lys-Pro-Cys-~la-H3s-Ahx-GI~-Cys-Ito-~la-Leu-OH
44. Ac-Asp-Lys-Pro-Cys-Ala-H3s-Ahx-Gly-Cys-llo-~la-Lou-OH
45. H-Asp-~ys-Pro-Cys-Ala-H1s-Ahx-Gly-Cys-llo-~la-Leu-NH2 46. Ac-Asp-Lys-Pro-Cy~-~la-His-~hx-Gly-Cys-~ls-Ala-Lau-NH2 ~ ;)5056 47 H-Asp-Lys-~ro-Hcy-Ala-His-Ahx-Gly-Cys-lle-Ala-Leu-OH
4~ Ac-Asp-Lys-Pro-Hcy-Ala-His-Ahx-Gly-Hcy-Ile-Ala-Leu-NH2 49 H-Asp-Lys-Pro-Cys-Ala-His-Ahx-Gly-Hcy-~le-Ata-Leu-OH
Ac-Asp-Lys-Pro-Cys-Ala-His-Ahx-Gly-Hcy-Ile-Ala-Leu-NH2 Sl H-Asp-Lys-Pro-Hcy-Ata-~s-Ahx-Gly-Hcy-Ile-Ala-Leu-OH
S2 Ac-Asp-Lys-Pro-Hcy-Ala-His-Ahx-Gly-Hcy-Ile-Ala-Leu-NH2 53 ~-Val-Arg-Ser-Ser-Ser-Arg-Thr-Pro-Ser-Asp-Lys-Pro-Cys-Ala-H1s-Ape-Gly-Cys-Ile-Ala-Leu-OH
54 H-Val-Arg-Ser-Ser-Ser-Arg-Thr-Pro-Ser-Asp-LyS-Pro-Cys-~la-His-Abs-Gly-Cys-Ile-Ala-~eu-OH
~-Val-Arg-Ser-Ser-Ser-Arg-Thr-Pro-Ser-Asp-Lys-Pro-Cys-~la-Hts-Ahx-Gty-CyS-Ite-Ata-Leu-OH
56 H-Asp-~ys-Cys-Val-Ala-H~s-Ape-Gly-Cjs-lle-~la-~eu-OH
I
57 Ac-Asp-Lys-Cys-Val-Ala-Hls-Apo-Gly-Cys-Ile-Ala-Leu-NH2 58 H-Asp-~ys-Pro-Cys-Ala-His-Ape-Gly-II~-Cys-Ata-Leu-OH
59 Ac-~sp-Lys-Pro-Cys-Ala-His-Ap--61y-Ile-Cys-~la-Leu-NH2 ~-~sp-Ly5-Cys-Val-~l--Ht5-~p~-Gly-Ilo-Cys-Ala-Lau-O~
61 Ac-Asp-~ys-Cys-~al-~la-His-~p~-Gly-lle-Cjs-~la-~eu-NH2 I
62 H-~sp-Ly~-pro-c~s-~la-Hts-ApJ-~la-c~s-lls-~la-L~u-oH
63 Ac-A~p-L~s-Pro-Cys-~ta-His-~p--~la-Cys-Ila-~la-~eu-HH2 64 H-Lou-~rg-Sor-Ser-S~r-Gln-Asn-S~r-Ser-~sp-Lys-Pro-CyS-~ta-Hts-Ap~-Gly-Cys-Ilo-~la-Leu-O*
H-Leu-Arg-S~r-S-r-S~r-Gln-Asn-Sor-Ser-ASp-LyS-Pro-Cys-Ala-His-~bs-Gly-Cys-lle-~la-Leu-OH
66 H-Leu-~rg-S-r-Ser-S~r-Gln-Asn-5er-5er-Asp-Lys-Pro-Cys-Ala-H1s-~hx 61y-Cys-lt~-Ala-Lsu-OH
67 H-Lys-Pro-Cys-~la-Hts-~hx-Gly-Cys-Ilo-~la-Leu-OH
68 H-Lys-Pro-Cys-Ata-~ts-~oc-Gly-C~s~ Al~-L~u-OH

- 24 - O.Z. 0050/40390 ExAMæLE 69 Ac-Pro-Oap-Ata-~is-Aoc-Gly-ASp-lle-Ala-Leu-~H2 1.02 g of Boc-Leu-MBHA-resin (~ubstitution 0.49 mmol/g), corresponding to a batch size of 0.5 mmol, were reacted a~ in AIa with 2 mmol each of Boc-Ala-OH Boc-Gly-OH Boc-Ala-OH
~oc-Ile-OH Boc-Aoc-OH Boc-Dap(Z)-OH
Boc-Asp-(OChx)-OH Boc-Hi8(Z)-OH ~oc-Pro-OH

(step~ 14-16 were dispen~ed with in ~11 the couplings following Hi~. After the ~ynthesi~ was complete, the N
terminu~ was acetylated (~tep~ 1-6 and 14-16 a~ in AI~.
The resulting peptide-re~in was dried under reduced pres~ure; tha yield was l.S8 g.

The crude product (385 mg) obtained after HF cleavage a~
in AII wa~ di~olved in 500 ml of degas~ed DMP, and 0.43 ml of triethylamine and, at -25-C, 0.43 ml of diphenylphosphoryl azide were added. The mixture was stirred at -25-C for 2 h, stored at -20-C for 2 day~, at 4C for 2 day~ and at roo~ temperature for 2 days, and 3ubsequently evapor~ted to dryne~s. The crude peptide was purified by gel chromatography (SEPHAD~X~-LH 20) and ~ub-~equ~nt medlum pre~ure c~romatography (cf. A IV, 50-70% A, 0.254 min1). 122 mg of pure product were obtained.

EXANP~E 70 Ac-~ys-Prcl-Oap-~la-~~1S-~hx-Gty-~sp-Ile-~la-Lou-NH2 1 g of resin dew ribed by Breipohl ~t al. (fram BASHE~), correspondinq to a batch size of 0.5 mmol, wa~ reacted a~ in AIb with 2 ~ol e~ch of 200505t~
- 25 - o.z. 0~50/4~39 Fmoc-Leu-OH Fmoc-Gly-OH Fmoc-DaptZ)-OH
Fmoc-Ala-OH Fmoc-Ahx-OH Fmoc-Pro-OH
Fmoc-Ile-OH Fmoc-His(Trt)-OH Fmoc-Ly~(Z)-OH
Fmoc-Asp(OtBu)-OH Fmoc-Ala-OH

After the synthesic wa~ completed, the M terminu~ was acetylated (steps 2-4 and 8-9 as in AIb). The peptide-resin was dried under reduced pressure; yield 1.75 g.

The crude product (518 mg) obtained after TFA cleavage as in AIII wa~ di~olved in 500 ml of degas~ed DMF. After addition of 0.43 ml of triethylamine and (at -25C~
0.43 ml of diphenylphosphoryl azide, the mixture was ~tirred at -25C for 2 h, and stored at -20C for 2 days, at 4C for 2 days and at room temperature for 2 days. It was then evaporated to dryness, and the crude peptide wa~
purified by gel chromatography (SEPHADEX~ LH 20). The i olated monomer (182 mg) wa~ deprotected with HF as in AII and purified by medium pressure chromatography (cf. AIV; 55-75% A; 0.25~ min~l). 144 mg of pure product were obtained Ac-Dap-Ala-His-Aoc-Glu-OH

6.45 g of F~oc-Glu(OtBu)-Merrifield resin (substitution O.31 mmol/g), corre~ponding to a batch size of 2 mmol, were reacted as in AIb with 8 mmol each o~

Fmoc-Aoc-O~ Fmoc-Ala-OH
Fmoc-Hi~(Tos)-OH Fmoc-Dap(Boc) OH

Subsequently, N-termin~l deprotection and acetylation were carried out (steps 2-4 and 8-9 as in AIb) and the t-butyl and Boc protective group~ were eliminated (~tep~
1-6 a~ in AIa). The cyclization on the reain took place Z11,30505~;
- 26 - O.Z. 0050/40390 in NMP with the addition of 3.54 g of BOP and 3.5 ml of dii~opropylethylamine (16 h). The peptide-resin was dried under reduced pressure. The yield was 7.0 g. The crude product obtained after HF cleavage a~ in AII was purified by gel filtration (SEPHADEX~ G-15) and medium pre~ure chromatography (cf. AIV; 5-20% A; 0.25% min~1). 21 mg of pure product were obtained.

The following can be prepared in a ~imilar manner to Examples 69, 70 and 71:

72. Ac-oap-Ala-H1s-Aoc-~sp-NH2 73. Ac-Oap-~la-Hi5-AOC-GlU~~Ha 74. Ac-Lys-~1a-His-Ano-Glu-NH2 75. H-L~s-~la-~is-~h~-G~u-OH
76. Ac-Lys-Ala-His-Ahx-61u-OH
77. H-Dap-~la-H1s-Aoe-Gtu-OH
78. Ac-oap-Ala-His-~d~-~sp-NH2 79. Ac-Asp-Ala-H~s-Aoc-Oap-NH2 ao . Ac-Glu-Ala-His-Ano-oap-NH 2 81 . AC-ASp-Al a-H~s-Ap~-Gly-Ljs-NH2 82. Ac-Lys-Al~-His-Ahx-Gly-Asp-NH2 ,_ ~
83 . AC-ASp-A 1 a-His-Leu-Gly-Oap-NH2 84. Ac-Pro-Asp-Ala-H1s-Apo-61y-Oap~ -NH2 85. Ac-Lys-Pro-Asp-Ala-His-Ape-Gly-Lys-Ile-Ala-NH2 86. Ac-Asp-Lys-Pro-Asp-Ala-His-Ape-Gly-Lys-lte-Ala-Leu-NH2 2'~30505~i - 27 - O.Z. 0050/40390 87 Ac-Asp-Lys-Pro-Asp-Ala-His-Ape-Gly-Lys-lle-Ala-Leu-OH
88 Ac-Asp-Lys-Pro-Glu-Ald-His-Ape-Gly-Lys-lle-Ala-Leu-NH2 -9 Ac-Asp-Lys-Pro-Glu-Ala-His-Ape-Gly-Lys-lle-Ala-Leu-OH
90. Ac-Asp-Lys-Pro-Lys-Ala-His-Ape-Gly-Asp-lle-Ala-Leu-NH2 91. Ac-Asp-Lys-pro-Lys-Ala-His-~pd-Gly-Asp-Ile-p~la-Leu-oH
92 H-Asp-Lys-Pro-Lys-Ala-His-Ahx-61y-Asp-ll~-Ala-L2u-NH2 93 Ac-Asp-~ys-Pro-Oap-Ala-His-~hx-Gly-Asp-lle-Ala-Leu-OH
94. Ac-Pro-~sp-Lys-Asp-~/al-~la-His-Ape-Gly-orn-lle-~ta-Leu-NH2 Ac-Pro-~sp-Lys-~sp-Yal-Ala-His-~pe-Gly-Orn-ll~-~la-Leu-OH
96 Ac-Asp-L~s-Pro-~sp-~la-His-~p~-Gly-ll~-Orn-~la-Leu-NH2 97 H-Asp-Lys-Pro-Asp-~la-His-Ap~-Gly-ll~-Orn-Ala-Leu-O~
_ 98 Ac-Pro-Asp-~ys-~sp-Val-Ala-His-Ape-Gly-ll~-Dap-Ala-~eu-NH2 99 Ac-Pro-Asp-Lys-Asp-Yal-Ala-His-Ap~-Gly~ -Dap-Ala-Leu-OH
100. H-Asp-~ys-Pro-Asp-~la-His-Ap~-~la-Oap-llo-Ala-~eu-NH2 101 Ac-Asp-~s-Pro-Asp-Ala-H~s-Ap--Ala-Oap-lle-Ala-L~u-OH ,

Claims (7)

1. A peptide of the formula I
X-Ala-His-A-Y I
where A is Val, Leu, Ile or -NH-(CH2)m-CO- (with m being an integer from 1 to 12), X is G-NH-CHM-CO-, G-NH-CHM-CO-W-, G-R-NH-CHM-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 R is , or a sequence of 5-11 amino acid residues from one of these peptide chains or a chain composed of 1-4 naturally occurring .alpha.-amino acids, U, V, and W are chains composed of 1-4 naturally occur-ring .alpha.-amino acids, and M and Q are hydrogens or one of the following -CH(CH3)2, -CH(CH3)-C2H5, -C6H5, -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-C8H5, HS, H2N, HO-CO, H2N-CO or H2N-C(=NH)-NH) or N and Q together are a -(CH2)e-S-S-(CH2)d-, -(CH2)e-CO-NH--(CH2)S- or -(CH2).-NH-CO-(CH2)g-NH-CO-(CH2)f- bridge (with c and d bcing from 1 to 4, e and f being from 1 to 6 and g being from 1 to l2), Fig.

as well as the salts thereof with physiologically tolerated 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-protective 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)e-NH-CO-(CH2)f- or -(CH2)e-NH-CO
(CH2)g-NH-CO-(CH2)f.

5. A peptide as claimed in claim 1 to 4 for use for controlling diseases.

6. The use of a peptide as claimed in claims 1 to 4 for controlling neoplastic diseases and autoimmune diseases as well as for controlling and preventing infections, inflammations and transplant rejection reactions.

7. A process for the preparation of a peptide as claimed in claims 1 to 4, which comprises preparation thereof using conventional methods of peptide chemistry.