CA2005051A1 - Tnf peptides - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE:

Peptides of the formula X-A-Gly-B-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

35051.

0. Z . 0050/40382 NOVEL TNF PEPTIDES

The present invention relates to novel peptides derived from tumor necrosis factor (TNF), the preparation thereof and the use thereof as drugs.

Carswell et al. (Proc. Natl. Acad. Sci. USA 72 (1975) 3666) reported that the serum of endotoxin-treated animal~ which had previously be~n infected with the Calmette-Guerin strain of Mycobacteria (BCG) brought about ~emorrhagic necrosi~ in various mou~e tumor~. Thi~
activity was ascribed to tumor necro~is factor. TNF al30 has a cytostatic or cytotoxic effect on a large number of transformed cell line~ in ~itro, wherea~ normal human and animal cell lines are unaffected ~LymphokinQ Reports Vol.

2, pp 235-275, Academic Press, New York, 1981). Recently, the biochemical characterization and the gene for human TNF have been de~cribed (Nature 312 (1984) 724, J. Biol.
Chem. 2fiO (1985J 2345, Nucl. Acid~ R~s. 13 (1985) 6361).

It is pos~ible to deduce from thi data the following protein ~tructure for mature human TNFs V~lK}~ ~f~es`~5~0~ Ly~Va~ Avalva~lah~
G~.~u~
ValGl~_I~V l~talP~GluGlyr_n~l~rSQr GlnV~1~aE~eLy ~ y~$aycy~6orDl~vniu3~euIh~i~dhrIle SEd~gIle~laVal;~dyrG~tllLy~V l,_~L~3dU~IleLy ~
CydDnknl~lhrPnXauGlyA~u~Lyuan~prnCiuPro~ Ieu q ~ l ~ t~S ~ ayGl~lValq ~ yIleI~

The TNF genes of cattle, rabbits and m~ce have al~o been described (Cold Spring Harbor Symp. Quant. Biol. 51 (1986) 597).

~eside its cytotoxic properties, TNF i~ one of the main ` 201~505~
- 2 - 0.Z. 0050/40382 sub~tances invol~ed in inflammatory reactions (Pharmac.
Res. 5 (1988) 129). Animal model~ have 8hown that TNF is involved in septic shock (Science 229 ~1985) 869) and graft-verYus-host disease (J. Exp. Med. 166 (1987) 1280).

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

The present invention relates to peptide.~ of the formula I

X-A-Gly-B-Y
where A i~ Asn, Asp or His, ~ i~ Val, Met or Phe, X i~ 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 i~ OH or NX2 or a carboxyl-protective group or G and Z together are al80 a covalent bond or -CO-(CH2).-NH- where a is from 1 to 12, R, U, V and W are peptide chain~ composed of 1-4 naturally occurring ~-amino acids, and M and Q are hydrogen~ or one-of the following -CH(CH3)2, -CH(CH3)-C~H~ t ~CaH" -CH(OH)-C~3, -CH2 ~ -cH2 ~ ~ or -(cH2)b-T
H H
(b being fro~ 1 to 6 ~nd T being hydrogen or OH, CH30, CH3S, (CH3)2CH, C8H5, p-HO-CeH~, HS, H2N, HO-CO, H2N-CO, H2N-C~=~H)-NH~ or M and Q togsther are a -(C~2)o-S-S-~CH2) d~ - ( CH2) ~-CO--NH-(CH2)s or -(CH2).-NH-CO-(CH2)~-NH-CO-(CH2)~-bridge (with c and d being from 1 to 4, e and 201350~;1.

3 o.z. 0050/40382 f being from 1 to 6 and g being from 1 to 12), as well a~ the ~alts thereof with physiologically toler-ated acids.

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

Particularly preferred phy~iologically tolerated acids are: hyorochloric acid, citric acid, tartaric acid, lactic acid, pho~phoric acid, methanesulfonic acid, acetic acid, formic acid, mzleic acid, fumaric acid, malic acid, ~uccinic acid, malonic acid, sulfuric acid~
L-glutamic acid, L-aspartic acid, p~ruvic acid, mucic acid, benzoic acid, glucuronic acid, oxalic acid, a3cor-bic acid and acetylglycine.

The novel peptides can be open-chain (G = H, amino-protective group; Z = OH, NH2, carboxyl-protective group, M and Q not connected together) and, in particular, have a disulfide brid~e (G = H, amino-protectlve group;
Z = OH, NH2, carboxyl-protective group; M + Q = -(CH2)C-S-S-(CH2)d-) or a ~ide chain br$dge (G = H, ~;no-protec-tivs group, Z = OH, ~H2~ carboxyl-protecti~e group, M ~ Q
= -(CH2).-NH-CO-(CH2)s~ or -~CH2).-NH-CO (CH2),-NH-CO-(CH2)r-) or be linked head-to-tail (G + Z - covalent bond or -CO-(CH2).-NH-).

The novel compound~ can be prepared by conventional method~ of pept~de chemistry.

Thus, the peptide~ can be constructed sequentially from amino acid~ or by linking tog~th~r suitable smaller peptide fragment~. In thQ 3equential c~nstruct$on, the peptide chaln i~ extended ~tepwi~ y one amino acid 20e~50~.

- 4 - O.Z. 0050/40382 each time, starting at the C terminus. In the ca~e of coupling of ~ragments it i~ possible to link ~ogether fragments of different lengths, these in turn baing obtainable by sequential construction from amino acid~ or coupling of other fragment~. The cyclic peptides are obtained, after synthe~is of the open-chain peptides, ~y a cyclization reaction carried out in high dilution.

In the case both of sequential con~truction and of fragment coupling it is neces~ary for the buildinq block~
to be linked by formation of an amide linkage. Enzymatic and chemical methods are ~uitable for this.

Chemical method~ for forming amide linkage~ are dealt with in detail by Nuller, Methoden der Organischen Chemie (Methods of Organic Chemi~try) Vol. ~V/2, pp 1-364, Thieme Verlag, Stuttgart, 1974; Stewart, ~oung, Solid Phase Peptide Synthesi~, pp 31-34, 71-82, Pierce Chemical Company, Rockford, 1984; Bodan3zky, Rlau~ner, Ondetti, Peptide Synthesis, pp 85-128, John Wiley & Son~, New York, 1976 and other ~tandard work~ of peptide chemi~try.
Particularly preferred are the azide method, the ~ymmetr-ical and mixed anhydride method, active e~ters generated in ~itu or preformed and the form~tion of amide linkage~
u~ing coupling reagents (activators), in p~rticular dicyclohexyl~arbodiimide (DCC), diisopropylcarbcdii~ide (DIC), l-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (~DQ), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (2DCI), n-propanepho~phonic anhydride (PPA), N,N-bis(2-oxo-3-oxazolidinyl)amidophosphoryl chloride (BOP-Cl), diphenylphosphoryl azide (DPPA), Ca~tro's reagent (~OP), O-benzotriazolyl-N,N,N',N'-tetra-methyluroniu~ ~alts (HBTU), 2,5-diphenyl-2,3-dihydro-3-oxo-4-hydroxythiophene dioxide (Steglich' B reagent;
HOTDO) and l,l'-carbonyldiimidazola (CDI). The coupling reagents can be amployed alonQ or in combination with additives ~uch A~ N,N~-dimethyl-4-aminopyridine (DMAP), ~:0~50~1.

- 5 - O.Z. ~050t40382 N-hydroxybenzotriazole (HOBt), N-hydroxybenzotriazine (HOOBt), N-hydroxysuccinimide (HOSu) or 2-hydroxy-pyridine.

Whereas it i~ normally po~ible to di~pense with protec-tive groups in enzymatic peptide synthesis, for chemical synthesis it is nece~sary for there to be xeversible protection of the reactive functional group~ which are not involved in the formation of the amide linkage on the two reactants. Three conventional protective group techniques are preferred f~r chemical p~ptide syntheses:
the benzyloxycarbonyl (z)~ the t-butyloxycarbonyl (Boc) and the 9-fluorenylmethyloxycarbonyl (Fmoc) techniques.
In each cas~ the protective group on the ~-amino group of the chain-extending building block is identified. The side-chain protective groups on the trifunctional amino acids are chosen 80 that they are not neces~arily elimin-ated together with the ~-amino protective group. A
detailed review of amino acid protective group~ i8 given by ~ller, Methoden der Organischen Chemie Vol XV/l, pp 20-906, Thieme Verlag, Stuttgart, 1974.

The building block~ used to construct the peptide chain can be reacted in solution, in suspension or by a method similar to that described by Merrifield in J. Amer. Chem.
Soc. 85 (1963) 2149. Particularly preferred methods are tho~e in which peptides are constructed sequentislly or by fragment coupling by use of the Z, ~oc or Fmoc protec-tiva group technique, in which case the reaction takes place in solution, as well as those in which, similar to the M~rrifield technique, one reactant i~ bound to an insoluble polymeric support (also called rQsin herein-after). This typically entails the peptide being con-~tructed sequentially on the polym~ric support, by use of the Boc or Fmoc protective group technique, wLth the growing peptide chaLn beLng covalently bonded ~t the C terminus to the insoluble resin particles ~c~. FLqures 20~50~1.

- 6 - O.Z. 0050/40382 1 and 2). This procedure allow~ reagents and byproducts to be removed by filtration, and thus recrystallization of intermediates is superfluous.

The protected amino acids can be bonded to any suitable polymers which merely need to be insoluble in the ~ol-vents used and to have a qtable phy~ical form which allow~ ea y filtration. The polymer mu~t contain a functional group to which the fir3t protected amino acid can be firmly linked by a covalent bond. A wide variety of polymers is suitable for thi~ purpo~e, for example cellulose, polyvinyl alcohol, polymethacxylate, ~ulfon-ated polystyrene, chloromethylated copolymer of styrene and divinylbanzene (Merrifield re~in), 4-methylbenz-hydrylamine-resin (MBHA-resin), phenylacetamidomethyl-resin (Pam-resin), p-benzyloxy~enzyl alcohol-re~in, benz-hydrylamine-resin (BHA-resin), 4-hydroxymethyl-benzoyloxymethyl-resin, the resin u~ed by Breipohl et al.
(Tetrahedron Lett. 28 (1987) 565; fr~m ~ACHEM), HYCRAM
rQsin (from ORPEGEN) or SASRIN resin (from BAC~EM).

Solvent~ suitable for peptide ~ynthesis in ~olution are all those which are inert under the reaction conditions, in particular water, N,N-dimethylformamide (DMP), dimethyl sulfoxida (DMS0), acetonitrile, dichloromethan~
(DCM), 1,4-dioxane, tetrahydrofuran (THF), N-methyl-2-pyrrolidone (NMP) and mixtures of the said solvents.Pept~de 3ynthe~is on polymeric support~ can be carried out in all inort organic solvents which di~olve the amino acid derivatives used; howev~r, solvents which also have re~in-swelling propertie~ are preferred, such a~
D~E, DCM, ~MP, aceton~trile and DMS0, a~ well as mixtures of these solvents.

After the peptide ha~ been synthe3ized it is cleaved off the poly~eric support. The cleava~e condit~on~ for the various type~ of rQsins are disclosed in the liter~ture.

- Z0~3~iO~l.
.

7 - O.Z. 0050/40382 The cleavage reaction~ mo~t commonly use acid and palladium catalysi~, in particular cleavage in anhydrous liquid hydrogen fluoride, in anhydrou~ trifluoromethane-sulfonic acid, in dilute or concentrated trifluoroacetic acid or palladium catalyzed cleavage in THF or THF-DCM
mixtures in the presence of a weak ba~e ~uch a3 morpho-line. The protective group~ may, depending on the choice thereof, be retained or likewi~e cleaved off under the cleavage conditions. Partial deprstection of the peptide may al~o be worthwhile if the intention i9 to carry ou~
certain deri~atization reactions or a cyclization.

Some of the novel peptide~ have good cytotoxic proper-ties. Some other~ of the peptide~ have high affinity for the cellular TNF receptor without, however, having cytotoxic activity. They are therefore T~F antagonists.
~hey compete with natural TNF for binding to the cellular TNF receptor and th~s suppress the TNF effect. The novel peptides are valuable drug~ which can be employed for treating neoplastic di~ease~ and autoimmune diseases as well as for control-ling and preventing infections, inflammations and tran~plant re~ection reactlon~. Simple experimentR can be u~ed to elucidate the mode of action of the individual peptides. The cytotoxicity of the peptide i~ determined by incubating a TNF-~ensitive cell line in the pre~ence of the peptide. In a second experi-mental approach, the cell line i~ incubated with the relevant peptide in the pre~ence of a lethal amount of TNF. It is po~ible in thi~ way to detect the $NF-antagon~tic effect. In addition, the aff~nity of the peptide for the cellular TNF receptor i~ determined in an in vitro binding experiment.

The ~ollowing te~t system~ were u~ed to characterize the agoni~tic and antagoni tic effect~ of the novel peptides:

I. Cytotoxicity test on ~N~-~ensitive indicator cells, 5051.

- 8 - O.Z. 0050/40382 II. Cytotoxicity antagonism test on TNF-sen~itive indicator cell~, III. Competitive receptor-binding test on indicator cells expressing ~NF receptor.

I. Cytotoxicity test The agonistic effects of the novel peptides are asses~ed on the basi~ of their cytotoxic effect on TNF-sensiti~e cell~ (e.g. L929, ~CF-7, A204, U937).
The test with L929 and MCF-7 wa3 carried out as followq:

1. 100 ~1 of culture medium containing 3 to 5 x 103 frs~hly trypsinized, exponentially growing, L929 cells (mouqe) or MCF-7 cell3 (human) were pipetted into the well~ of a 96-well 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.

The L929 culture mediu~ contained 500 ml of lx ~arle'~ MEM (Boehringer Mannheim), 50 ml of heat-inactivated (56C, 30 min) fetal calf serum (FCS), 50 ml of L-glutamine (200 mM), 5 ml of lOOx non-essential amino acids, 3 ml cf lM HEP~S
buffer pH 7.2,and 50 ml of gentamicin (50 mg/ml).

The ~CF-7 culture medLu~ contained 500 ml of lx Dulbecco'~ NEN (Boehringer MRnnheim~, 100 ml of heat-inactivated (56-C, 30 min) FCS, 5 ml of L-glutaMine and S ml of lOOx non-es~ential amino acid~.

2. Th~ nex~ day 100 ~1 of the peptide ~olution to be tested were added to the cell culture8 and ~ub~ec~ed to serial 2-fold dilution. In addition, some cell control~ (i. 8 . coll cultures not 50~.

g o.Z. 0050/40382 treated with peptide dilution) and ~ome rhu-TNF
controls (i.e. cell culture~ treated with recom-binant human ~NF) were al~o made up. The culture plate was incuba~ed at 37C in an atmoc?here of air saturated with water vapor and containing 5 C02 by volume for 48 h.

3. The percentage of 3urviving cells in the cultures treated with peptide dilution wa~ determined by staining with cry~tal violet, For thi~ purpose, the liquid was removed from the well~ of the test plate by tapping it. 50 ~l of cry~tal violet solution were pipetted into aach well.

The composition of the ~ry~tal violet solution was a~ follows:

3.75 g of crystal violet 1.75 g of NaCl 161.5 ml of sthanol 43.2 ml of 37% formaldehyde water ad 500 ml The cry~tal violet solution wa~ left in the w~118 for 20 min and then likewise remov~d by tapping.
The plate~ were then washed 5 times by immer~ion in water in order to remove dye not bound to the cell~. The dye bound to the cell~ Wafi extracted by adding 100 ~l of reagent solution ( so~ etha-ncl, 0.1~ glacial acetic acid, 49.9% water) to each well.

4. The plates were ~haken for 5 min to obtain a ~olution of unifor~ co~or in each woll. ~he ~urviving cQll~ were det~rmined by measuring ~he extinction at 540 nm of the colored solution in the individual well~.

210~0.~

- 10 - O. Z . 0050/4D382 5. Subsequently, by relating to the cell control, the 50% cytotoxicity value wa~ defined, and the reciprocal of the sample dilution which re~ulted in 5096 cytotoxicity wa~ calculated as the cyto-toxic activity of the test sample.

II. Cytotoxicity antagonism te~t The antagoni~tic effect of the peptides wa~ as~essed on the basis of their property of antagonizing the cytotoxic ef fect of rhu-l'NF on lq~ en~itive cells (e.g. L929, MCF-7, A204, U937). The cytotoxicity antaqonism test with L929 and ~CF-7 cells wa~
carried out a~ follow~ s 1. 100 ~1 of culture medium containing 3 to 5 x 103 fre~hly tryp~inized, exponentially growing, L929 cells (mou~e) or NCF-7 cells ( human) were pipetted into the wells of a 96-well flat-bottom culture plate . The plate was incubated at 37 C
overnight. ~he air in the incubator wa~ saturated with wat0r vapor and contained 5% CO2 by volume.

2û The h929 culture medium cont~ined 500 ml of lx Earle'~ MEN (Boehringer Nannhei~), 50 ml of heat-inact~vated (56DC, 30 min) PCS, 5 ml of I.-gluta-mine ( 200 mN), S ml o~ 100~ non-e~Qntial amino acid~, 3 ml of lM HEPES buffer pH 7.2, and 500 ~1 of gentamicin (50 mg/ml).

The MCF-7 culture medium contained 500 ~1 of lx Dulbecc0'8 MEN (Boehringer Msnnheim), 100 ml of hea~-in~ctivated (56-C, 30 min) FCS, 5 ml of h-glut~mine (200 mM) and S ml of lOOx non-essQn-tial amino acid~.

2. The next day 100 ~1 of the pe~tide solution to be to~tQd w~re added to the cell cultures and 7~0~505~

O.Z. 0050~40332 ~ub~ected to serial 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 addition, ~ome cell controls (i.e. cell cultures not treated with peptide solution or with rhu-TNF solution) and so~e rhu-TNF controls (= cell cultures treated only with rhu-TNF solution) were also made up. The culture plate wa~ then incubated at 37~C in an atmo~phere of air ~aturated with water vapor and containing 5% CO2 by volume for 48 h.

3. The percentage of surviving cell~ in the culture~
treated with substance solution wa~ determined by staining with cry~tal violet. Por thi~ purpose, the liquid wa~ removed from the wells of the test plate by tapping it. 50 ~1 of cry~tal violet ~olution were pipetted into each well.

The crystal violet solution had the composition specified in I.3 The crystal violet solution wa~ left in the wells for 20 min and then likewi~e removed by tapping.
The plates were then wa~hed 5 tim~s by immersion in water in order to remove dye not bound to the cells. The dye bound to the cells wa~ extracted by adding 100 ~1 of reagQnt solution (50% etha-nol, 0.1% glacial acetic acid, 49.9~ water) to each well.

4. The plates were 3h~ken for 5 min to obtain a ~olution of uniform color in each wall. The sur~iving cells were determin~d by mea~uring the extinction at 540 nm of the colored solution in the ~ndividual well8 .

~o~l~35~
`\
- 12 - O.Z. 0050/40382 5. Subsequently, by relating to the cell control and the rhu-TNF control, the 50% antagonism value wa~
defined, and the ~ample concentration which resulted in 50~ antagonism of rhu-TNF cytotox-icity at the rhu-TNF concentration used was calculated as antagonistic activity of the sample tested.

III. Competitive receptor-binding test Both the agonistic and antagoni~tic effects of peptides ars conditional on the latter binding to the TNF receptor. Thi~ mean~ that peptides with an agoni~tic or antagonistic effect compete with rhu-TNF for binding to the TNF receptor on TNF-sensitive indicator cell le.g. U937). The competi-tive receptor-binding test was carried out as follo~s:

1. 100 ~1 of medium containing various concentra-tions of the peptide to be tested and of rhu-TNF
(= control) were pipetted into the reaction ve~els. The mediu~ compr~ed 500 ml of ~8S
(Boehringer ~annheim) I 10 ml of he~t-inactivated (56-C, 30 min) FCS and lO0 mg of sodium azide.

2. Sub~equently, lO0 ~l of medium containing 1 ng of ~ labeled rhu-TN~ ( Bolton lactoperoxida~e method) were placed in the reaction ~e~sels and mixed. The non-specific bindin~ (NSB) WR~ deter-mined by ~ixing in the reaction ve~els the ~25I-labeled rhu-TNF (l ng of l2~I-rhu-TNF in lO0 ~l of medium) with a 200-fold exce3s of unlab0led rhu-TNF (200 ng of rhu-TNF in 100 ~1 of msdium).

3. Then lO0 ~l of medium containing 2 x 106 U937 cell~ (human) w~re pipetted into the reaction .

2~l~5o5l - 13 - O.Z. 0050/40382 ves~el~ and mixed. The reaction ves~els ~test volume 300 ~l) were incubated at 0C for 90 min.
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 time~
with medium and transferred quantitatively into coun~ing vial3, and the cell-bound radioactivity wa~ determined in a Clini gamma counter 1272 (LKB Wallac).

5. After the measurement~ had been corrected for the non-~pecific binding, the 50% competition value was defined by relation to the overall binding, and the sample concentration which led to 50%
competition of125I-rhu-TNF binding at the12sI-rhu-T~F concentration used was calculated a~ the co~petitive activity of the ~ample tested.

The Examples which follow are intended to explain the invention in more detail. The proteinogenou~ amino acids are abbreviated in the Exa~pla~ u~ing the conventional three-lettar code. Other meaning~ ares Aad - ~-aminoadipic acid, Abs = 4-aminobutyric acid, A~
= a~etio acid, Aoc = 8-aminoo~tanoic acid, APQ - 5-aminopentanoic acid, Hcy = homocy~teine, Hly = homo-ly8in6, Orn ~ ornithine, Dap = 2,3-diaminopropionic acid.

A. General procedure I. The peptide~ claimed in claim 1 were synthe~ized usinq standard methods of ~olid-phase peptide synthesi~ in a completely Automatic model 4~0A
peptide ~ynth~izer from APPLIED 8IOSYS~EMS. The apparatus u~es differen~ synthe~i~ cycle~ for the Boc ~nd F~oc protective group technique~.

zo~sos~

- 14 - O. Z . 0050/4~382 a~ Synthesis cycle for the Boc protecti~e group technique 1. 30% trifluor~etic acid in DCM 1 x 3 min 2. 50% trifl~cctic æid in DCM 1 x 17 min 3. DCM washir~ 5 x 1 min 4. 5% diisc~let~le~nine in DCM 1 x 1 min 5. 596 dii~leth~lamire in ~P 1 x l min 6. ~P washing S x 1 min 7. A~dition of ~iVatf~ p~ed am~
acid (activa'cion ~y 1 ff3~ivalent of D~
and 1 equivalent of E~Bt in ~P/DQq);
peptide ca~pliny (l~t part) 1 x 30 min 8. Ac3~itioTI of 1~;0 to the r~acti~n mixhlre it co¢~ 20% ~;0 ~y v~lu~

9. Peptide ca~ling (2nd part) 1 x 16 min 10. ~ition of 3.8 eq~ivalentç3 of dii~

11. Pepti~e cc~uplin~ (3rd part) 1 x 7 min 12. OCM w~hir~ 3 x l min 13. If reæti~ is inc~Qlete, ~itian of c~upling (re~n to 5. ) 14. 10% ~c a~ide, 5% dilsc~l-e~lanir.P in DCM 1 x 2 min 15. 10% æetic ar~ide in DQI 1 x 4 min 16. DQlwo~ing 4 x 1 min 17 }~turn to l.

lc~ NIP wo~ng 1 x 1 min 2. 20% piE~ri~ii~ in N~P 1 x 4 min 3. 20S pi~ridi~ in ~P 1 x 16 min 4. NMP wo~hir~ S x l min 5. h~iti~n of pL~ tivated p~t~l amino æid (a~:tivati~ ~y l ff~i~ral~t of DaC

p~ lQ c~ling l x 61 min 6. ~MP ~i~g 3 x l min 0~

- 15 - O.Z. 0050/40382 7. If rff~inn is ~xxoplete, n~ition of ~p~g (~ to 5.) 8. 10% a~etic ar~ride in 2~P 1 x 8 mir~
9. ~MP w hing 3 x 1 min lO.Return to 2.

II. Working up of peptide-re~ins obtained a~ in Ia ~he peptide-resin obtained as in Ia was dried under reduced pressure and tran~ferred into a reaction ve~sel of a Teflon HF apparatus (from PENINSULA).
Addition of a scavenger, preferably ani~ole (l ml/g of resin), and of a thiol, in the case of tryptophan-containing peptide~, to remove the indole formyl group, preferably ethanedithiol (0.5 ml/g of resin)~
was followed by condensation in of hydrogen fluoride (lO ml/g of re~in) while cooling with liquid N2. Ths mixture was allowed to warm to 0C, and wa~ Ytirred at this temperature for 45 min. The hydrogen fluor-ide was then stripped off under reduced pre3~ure and the residue was washed with ethyl acetate in order to remove re~aining scavenger. The peptide wa3 extracted with 30% strength acetic acid and filtered, and the filtrate wa~ freeze-dried.
To prepare peptide hydrazide~, the peptide-re~in (Pam- or Merrifield resin) was suspended in DMF
(15 ml/g of resin), hydrazine hydrate (20 equiva-lents) wa~ added, and the mi~ture was st~rred at room temperature for 2 d~ys. To work up, the re~in wa~ filtered off and the filtrate wa~ evaporated to ~ryness. The re~idue was cry~tallized from DMF/~t20 or NeOH/~2O-III. Working up of the peptide-resin~ obtained a~ in Ib The p~ptide-r~in obtained as in Ib wa~ dried under reduced pre~sure and ~ub~equently sub~ected to one of the following cleavage procedures, depending on tha amino acid composition (wade~ Tregear, Howard 2,0C~505~

- 16 - O. Z . O0S0/~0382 Florey E~noc-Workshop Manual, Melbourne 1985 ) .

~ SO~l - 17 - O.Z. 0050/40382 Peptide containing Cleavage conditions Arg(Mtr) Met Trp TFA Scavenger Reaction Time S
no no no 9S% S% H2O 1,S h yes no no 95% 5% thioani~ole 2 3 h no yes no 95% 5~ ethyl methyl 1.5 h 9ul fide no no ye~ 95~ 5~ ethanedithiol/ l.S h anisole ( 1: 3 ) no ye~ yes 95% 5% ethanedithiol/ 1.5 h anisole/ethyl methyl sulfide (1:3:1) ye~ yes yes 93% 7% ethanedithiol/ ~ 3 h anisole/ethyl methyl sulfide (1:3:3) The su~pen~ion of the peptide-resin in the ~uitable TFA ~ixture wa~ stirred at roo~ temperature for the stated time and then the resin wa~ filtered off and wa~hed with TFA and with DCM. The filtrate and the wa~hings were extensively concentrated, and the peptido was precipitated by addition of die~hyl ether. The mixture was cooled in an ice bath, and the precipitate wa~ filtere~ off, taken up in 30 acetic acid and free~e-dried.

IV. Purification and characterization of the peptide~
Purification was by gel chromatography (SEPHADEX
G-10, G-lS/10~ HQAc; S~PHAD~X LH20/MeOH) and ~ub-eequent medium pre~sure chromatography (stat~onary pha~e: HD-SIL C-18, 20-45 ~, lo0A mobil~ pha~ez gradient with A = 0.1% T~A/M~OH, B z 0.1% TFA/H2O).

Z0~3S05~

- 18 - O.Z. 0050J40382 The purity of the final products wa~ determined by analytical HPLC (stat~onary phase. 100 x 2.1 mm VYDAC C-18, 5 ~, 300 A; mobile phase = CH3CN/H20 gradient buffere~ with 0.1~ TFA, 40C). Charac-terization wa~ by mean~ of amino acid analy~is and fast atom bo~bardment ma3s spectromet~y.

B. Specific procedure~

H-Ala-Asn-Gly-Val-&lu-NH2 1.28 g of Boc-Glu(OBzl)-MBHA-re~in (~ubstitution 0.39 mmol/g), corre~ponding to a batch size of 0.5 mmol, were reacted a~ in AIa with 2 mmol each of Boc-Val-OH
Boc-Gly-OH
Boc-Asn-OH
Boc-Ala-OH.

After the synthesi~ was complete, the peptide-resin underwent N-terminal depro~ection (~tepff 1-3 as ln AIa) and subsequent drylng under reduced pre~sure; the yield was 1.35 g.

0.7 g of the resin obtained in thi~ way wa~ sub~ected to H~ cl~av~g~ as in ~II. The crude product (81 mg) was purifisd by gel filtration (SEP~AD~X G-10) and medium pre~sure chromatography (cf. AIV; 40-60S A; 0.25% min~l).

EXANPL~ 2 A~-Leu-Ala-Asn-Gly-Val-Glu-OH

O.46 g of Fmoc-Glu(Ot~u)-p-~lkoxybenzyl alcohol-rQsin (~ubstitution 0.55 mmol/g) corre~ponding to a batch ~i~e of 0.25 mmol, wa~ reacted a~ in AIb with 1 mmol ~sch of 20~0~

- 19 - O. Z . 0050/40382 E~noc-Val-OH Fm~c-Ala-OH
E moc-Gly-OH Fmoc-Leu-OH
Fmoc-Asn-OH

After the synthe~is was complete, the N terminus was acetylated ~steps 2-4 and 8-9 as in AIb). ~he resulting peptide-re3in wa~ dried under reduced pressure; the yield was 0.54 g.

The crude peptide (129 mg) obtained after TFA cleavag~ as in AIII was purified by gel filtration (SEPHADEX G-10) and medium pressure chromatography (cf. AIV; 40-60% ~;
0.25% min~1). 67 mg of pure product were obtained.

The following were prepared in a similar mann~r to Examples 1 and 2s 3 H-Ala-Asn-Gly-Val-Glu-OH

4 Ac-Ala-Asn-Gty-Val-Gtu-OH

Ac-Ata-Asn-Gly-val-Glu-NH2 6 H-Leu-~la-Asn-Gty-Val-Glu-OH

7 H-Leu-Ala-Asn-Gly-~al-Glu-NH2 8. Ac-Leu-Ala-Asn-Gly-Val-l;lu-NH2 9 ~-L~u-Ala-~sn-Gly-Val-Glu-Leu-O~
Ac-Leu-Ala-Asn-Gly-Val-Glu-Leu-OH

Il. H-Lou-Ala-Asn-Gly-Val-Glu-L~u-NH2 12 Ac-Leu-Ala-Asn-Gly-Val-Glu-Leu-NH2 13 H-Leu-Leu-Ala-Asn-Gly-Val-Glu-Leu-Arg-OH
14 Ac-Leu-Leu-~la-~sn-Gly-Val-Glu-Leu-~rg-OH
H-Lcu-Leu-~la-Asn-Gly-V~l-Glu-Leu-~rg-NH2 16 ~c-Leu-Leu-Ala-~sn-Gly-Val-Glu-Leu-~rg-NH2 17 H-~la-Leu-Leu-Ala-~sn-Gly-Val-Glu-Lou-~rg-OH
18 Ac-Ala-Leu-Leu-~la-Asn-~ly-Val-Glu-L~u-Arg-NH2 19 H-Leu-Ala-Asn-61~-Ph~-Glu-OH
Ac-~eu-Ala-Asn-Gly-Ph~-Glu-NH2 21 Ac-L~u-Ala-Asn-Gty-M~t-Gtu~Leu-NH2 22 Ae-~ou-L~u-~t~-H~s-Gly-Val-Glu-Lau-~rg-NH2~
23 ~e-1t~ sn-6ty-Val-Glu-NH2 ~c -Cy5 -Ala-A~n-Gly-Val -Glu-Cy8 -NH2 ~lO~O~.

- 20 - O.Z. 0050/40382 O.57 g of Boc-Cy~(pMB)-MB~A-resin (~ubstitution O.86 mmolJg), corre~ponding to a batch size of 0.5 mmol, was reacted a~ in AIa with 2 mmol each of Boc-Glu(OBzl)-OH Boc-A~n-OH
Boc-Val-OH Boc-Ala-OH
Boc-Gly-OH Bo~-Cys(pMB)-OH.

After the synthesi~ was cemplete, the N terminus was acetylated (steps 1-6 and 14-16 as in AIa).

The resulting peptide-re~in was dried under reduced pressure; the yield was 0.98 g.

0.49 g of the re~in obtained in this way wa~ ~ub~ected to EF cleavage as in AII. The freeze-dried crude product was taken up in 2 1 of 0.1% strength acetic acid, and the pH
wa~ then ad~usted to 8.4 with aqueous ammonia. Under an argon atmosphere, 0.01 N g3tFe(CN)~] solution wa~ 8 lowly added dropwise until the yellowi~h-green color per~i~ted for at least 15 min. The mixture was then ~tirred for 1 h and then acidified to pH 4.S with glacial acetic acid, and 15 ml of an aqueous suspen~ion of an anion exchanger (BIORAD~ 3 x 4A, chloride form) werQ added. After 30 min, the ion exchanger resin wa~ filtered off, and the filt-rate was concentrated to lOO ml in a rotary evaporator and sub~equently freeze-dried.

All the ~olvents used had previou~ly been saturated with nitrogen in order to prevant any oxidation of the free cy3teine residues.

The crude product was purified by gel chromatography (SEPHADEX~ G-15). 87 mg of pure product were obtained.

The following can be prepared in a similar manner to Rxample 24 (Pam-re~in was usQd to prepare the peptide 20ai5051 - 21 - O. Z . 005~/403~2 i acid~): 25. Ac-cys-Asn-Gly-val-cys-NH2 r-26. Ac-Cys-Ala-Asn-Gly-Val-Cys-NH2 2~. H-Cys-Ala-Asn-Gly-Val-Cys-OH
28. H-CyS-Ala-Asn-Gty-Val-Glu-Cys-OH

29. Ac-Cys-~la-Asn-Gly-~al-Glu-Cys-OH
r - i 30. H-Cys-~la-Asn-61y-Val-Glu-CyS-NH2 r-31. Ac-Cys-Asn-Gly-Val-61u-Cys-NH2 32. H-Hcy-Ala-Asn-Gly-Val-Glu-Cys-OH
33. Ac-Hc~-~la-~sn-Gly-Val-~lu-Cys-OH
__ i 34. H-Hcy-Ala-~sn-Gly-Vdl-Glu-Cys-NH2 35. ~c-Hcy-Ala-~srl-Gly-Val-Glu-Cys-NH2 r 36. H-Cys-~la-~n-61y-Val-61u-Hc~-OH
, 37. Ac-Cys-Ala-Asn-Gly-Val-Glu-Hcy-OH
38. H-Cys-Ala-Asn-Gly-Val-Glu-HCy-NH2 39. Ac-Cys-Ala-Asn-Gly-Val-Glu-Hcy-NH2 i 40. H-Hcy-Ala-Asn-Gly-Val-Glu-Hcy-OH
41. Ac-Hcy-Ala-ASn-Gly-Val-Glu-Hcy-OH

42. H-Hcy-AIa-Asn-Gly-Val-GIu-Hcy-NH2 r 43 . Ac-Hcy-AI a-Asn-Gly-Val-Glu-Hcy-NH2 44. H-Cys-Leu-Ala-Asn-Gly-val-Glu-Cys-OH
45. Ac-Cys-Leu-Ala-Asn-Gly-Val-Glu-Cys-NH2 46. H-Hcy-Leu-Ala-Asn-Gly-Val-Glu-Cys-OH
-4~. Ac-Hcy-~eu-Ala-Asn-Gly-Val-Glu-Cys-NH2 i 48. H-Cys-Ala-Asn-Gly-Val-Glu-Leu-Cys-OH
49. Ac-Cys-Ala-Asn-Gly-Val-Glu-Leu-Cys-NH2 r 50. H-Hcy-~la-Asn-Gly-Val-Glu-L~u-Cys-OH
r _ j 51. Ac-Hcy-Ala-Asn-Gl~-Yal-Glu-Lou-Cys-NH2 zo~so~

- 22 - O.~. ~050/40382 52. H-Cys-Leu-Ala-Asn-Gly-Val-Glu-Leu-Cys-OH

53. Ac-Cys-Leu-AIa-Asn-GIy-Val-GIu-Leu-Cys-NH2 54. H-Hcy-Leu-AIa-ASn-GIy-Val-GIu-Leu-Cys-OH
55. Ac-Hcy-Leu-AIa-Asn-GIy-Val-GIu-Leu-Cys-NH2 56. H-Cys-~eu-Ala-Asn-Gly-Val-Glu-Leu-Hcy-O~
57. Ac-Cys-Leu-Ala-Asn-Gly-Val-Glu-Leu-Hcy-NH2 58. H-Hcy-Leu-Ala-Asn-Gly-Val-Glu-Leu-Hcy-OH
59. Ac-Hcy-Leu-Ala-Asn-Gly-Val-Glu-~eu-~cy-NH2 60. Ac-Asn-Ala-Leu-Hcy-Ala-Asn-Gly-Val-Glu-Cys-Arg-Asp-NH2 61. Ac-Hcy-AIa-Asp-Gly-Val-Glu-Cys-NH2 62. H-Hcy-AId-Asn-GIy-Val-GIu-Hcy-OH

63. H-Hcy-Ala-His-Gly-Val-Glu-Cys-OH
~, 64. H-Cys-Leu-Ala-Asn-Gly-Val-Asp-Leu-Cys-OH
55. H-Hcy-Ala-Asn-Gly-Val-Cys-OH
i 66. Ac-Hcy-AIa-Asn-Gty-vat-Cys-NH2 67. H-Cys-Ala-Asn-Gly-Val-Hcy-OH
68. Ac-Cys-Ala-Asn-Gly-Val-Hcy-NH2 69. H-Hcy-~la-~Sn-Gty-Val-HCy-OH
70. ~c-Hcy-~ Asn-Gly-val-~c~-~H2 EaiUqP ~ 71 Ac-Lys-Ala-~sn-Gly-Val-Glu-NH2 0.53 g of ~oc-Glu(OChx)-MBHA-resin (~u~titution 0.95 mmol/g), corrssponding to a batch ~ize of 0.5 mmol, wss reacted a~ Ln AIa with 2 rmnol each of Boc-Val-OH Boc-Ala-O}~
Boc-Gly-OH ~oc-Lys(Cl-Z-)-OH
Boc-Asn-O~

20~5051 - 23 - O.Z. 0050/40382 After the synthesis was complete, the N terminus was acetylated (steps 1-6 and 14-16 as in AIa). The resulting peptide-resin was dried under reduced pressure; the yield was O . 86 g .

The crude product (272 mg) obtained after HF cleavage as in AII was dissolved in 380 ml of degassed DMF, and 0.53 ml of triethylamine and, at -25DC, 0.54 ml of diphenyl-phosphoryl azide were added. The mixture was stirred at -25C for 2 h, stored at -25C for 2 day~, at 4C for 2 days and at room temperat~re for 2 days and subsequently evaported to dryness. The crude peptide wa~ purified by gel chromatography (SEPHADEX~ G-15) and subsequent medium pressure chromatography (cf. AIV; 5-25~ A; 0.25~ min1).
51 mg of pure product were obtained.

Ac-Ly~-Ala-A~n-Gly-Val-Glu-Glu-NH2 2 g of resin described by Breipohl et al. ~from BACHEM), corresponding to a batch ~ize of 1 mmol, was reacted as in AIb with 4 mmol each of Pmoc-Glu(OtBu)-OH Pmoc-Asn-OH
Fmoc-Glut08zl)-OH Fmoc-Ala-OH
Fmoc-Val-OH Pmoc-Ly~(Boc)-OH
Fmoc-Gly-OH

25 After the synthe~is wa~ complete, the N t~rminu~ was acetylated (steps 2-4 and 8-9 a~ in AIb). ~he peptide-resin wa~ dried under reduced pre~sure; yield 2.71 g.

480 mg of the crude product (872 mq~ obtained after TPA
cleavage as in AIII were di~olved in 500 ml of degas~ed DMF, and 0.67 ml of triethyla~ine and, at -25C, 0.67 ml of diphenylpho~phoryl azide were added.The mixture was f~:O~S051.

- 24 - O.~. 0050/40382 stirred at -25C for 2 h, stored at -25C for 2 day~, at 4C for 2 days and at room temperature for 2 day~ and sub~equently evaporated to dryness. The crude peptide was purified by gel chromatography (SEPHADEXs LH 20) and the isolated monomer ~103 mg) wa~ deprotected with HF as in AII and purified by medium pre~sure chromatography (cf.
AIV, 5-25~ A, 0.25% min~~). 68 mg of pure product were obtained.

, - - ~
H-Lys-Leu-Ala-Asn-Gly-Val-Glu-Glu-OH

1.04 g of Fmoc-Glu(OtBu)-Merrifield resin ~ ub~titution O.48 mmol/g), corresponding to a batch ~iza of 0.5 mmol, were reacted a~ in AIb with 2 mmol each of Fmoc-Glu(08~ OH Fmoc-Asn-OH Fmoc-Lys(Boc)-OH
Fmoc-~al-OH Fmoc-Ala-OH
Fmoc-Gly-OH Fmoc-Leu-OH

The t-butyl and Boc protective groups were ~ub~equently cleaved off (steps 1-6 as in ~Ia). The cyclization on the re~in took place in NMP with the addition of 0.8g g of BOP and 0.87 ml of diisopropylethylamine (24 h). The peptid~-re~n underwent N terminal deprotection (~tep~ 2-4 a~ in AIb) ~nd drying under reduced pressure. ~he yield wa~ 1.3S g.

The crude product obtained after HF cleavage as in AII
wa~ purified by gel filtration (Sephadex G-15) and medium pre~sure chromatography (cf. AIV; 10-30% A; 0.25~ min~1).
24 mg of pure product wsre obtained.

The following can be prepared in a ~imilar manner to Examples 71, 72 and 73:

50~

- 25 - O . Z . 0050/40382 74. Ac-Aa~-Asn-Gly-Val-LYS-NH2 75. U-Gtu-Ala-Asn-Gly-Val-Lys-OH
76. Ac-Glu-Ala-Asn-Gly-Val-Ljs-NH2 77. bc-Glu-Ala-Asn-Gly-Val-Lys-OH
18. Ac-Lys-Asn-Gly-Val-Giu-NH2 79. Ac-Lys-ASn-Gly-Val-Glu-OH
80. Ac-Glu-Ala-Asn-Gly-Val-Glu-Hty-NH2 81. Ac-Asp-Ala-Asn-Gly-Val-Glu-Lys-NH2 82. Ac-Asp-Ala-Asn-Gly-Val-Glu-Lys-OH
83. Ac-Asp-Ala-Asn-61y-Val-Gtu-Hly-NH2 84. Ac-Orn-Ala-Asn-Gly-Val-Glu-~sp-NH2 85. Ac-Lys-Ala-Asn-61y-Val-Glu-OH
6. Ac-Lys-Ala-Asn-&ly-Val-Glu-~sp-NH2 _ -- 7 7. Ac-~gs-~l~-Asn-Gly-Val-Glu-Glu-OH
. Ac-Lys-Ala-Asn-Gly-Val-Glu-Aad-NH2 9. Ac-Hiy-Ala-A5n-61y-Val-Glu-A5p-NH2 0. Ac-Aad-~l a-ASn-Gly-Val-Glu-Hly-NH 2 1. Ac-~ad-~la-~sn-Gly-Val-Glu-Lys-NH2 . _ .
92. Ac-Lys-AI~ sn-61y-V-I-Gtu-NH2 93. Ac-Giu-Ala-Asn-Gly-Val-Glu-Leu-Ly5-NHl 94. Ac-Lys-Leu-Ala-~sn-Gly-Val-Glu-Glu-NH2 95. Ac-Glu-Ala-Asn-Gly-Val-Gtu-Leu-Lxs-NH2 -96. Ac-Lys-Ala-Asn-Gly-Val-Glu-Leu-Glu-NH2 97. H-Asn-Ala-Leu-Glu-Ala-Asn-Gly-Val-~ys-Leu-NH2 98. Ac-G~u-Gly-Asn-Gly-Val-Lys-NH2 99. Ac-Ala-L~u-Glu-~)a-~sn-Gly-Val-Glu-L~s-Arg ASp-NH2 - ~0~50~1.

- 26 - O.Z. 005~/40382 I 00 . Ac - A s p - A I a-H i s-G I y - V a I -G I u Ly S-NH 2 101. Ac-~ys-Ala-Asn-Gly-Phe-Gtu-Giu-NH2 102. Ac-Glu-Ala-Asn-GIy-Val-Glu-~ys-NH2 EXA~P~E 103 Leu-Ala-A~n-Gly-~al-Glu-Leu-Abs 1.47 g of Fmoc-Gly-p-alkoxybenzyl alcohol-resin (sub-stitution 0.68 mmol/g), corre~ponding to a batch size of1 mmol, were reacted a~ in Alb with 4 mmol each of Fmoc-A3n-OH Fmoc-Leu-OH
Fmoc-Ala-OH Fmo~-Glu(OBzl)-OH
Fmoc-Leu-OH Fmoc-Val-OH
Fmoc-Abs-OH

After the synthesis wa~ complete, the peptide-re~in underwent N-terminal deprotection (~tep~ 2-4 as in AIb) and ~ub~eguent drying under reduced pr~ura. The yield was 1.3S g.

200 mg of the crude peptide obtai~ed after ~FA cleavage a~ in AIII were di~301ved in 200 ml of degas~ed DMF. 84 mg of NaHC03 and 264 mg of BOP were added and then the mixture wa~ stirred at room temperature overnight. It was then evaporated to dryne~s, and the crude peptide wa~
purified by gel chromntography (SEPHAD~X LH 20). The isolated monomer (74 mg) was deprotected with HF a3 in AII. 62 mg of pure product were obtained.

The following can be prep~red in a similar manner to Example 103:

~0~05~

- 27 - O . 2 . 0050/40~82 104. rAta-Asn-GIy-Val-GIu 105. rLeu-AIa-Asn-GIy-Val-GIu-Leu-Arg 106. rAoc-Ala-Asn-GIy-Val-GIu 107. rAld-Asn-GIy-Val-GIu-Leu-Ape 108. rLeu-AIa-Asn-~ly-Val-Glu-Leu-Ape 109.rLeu-Ser-Asn-Gly-Val-Glul i 110.rLeu-Ala-~is-Gly-Val-Glu-Leu-Arg 111.rAla-Asn-Gly-Met-Glu-Leu-Apel tl2. rLeu-AIa-Asn-GIy-Val-GIu-l o

Claims (8)

1. A peptide of the formula I

X-A-Gly-B-Y I
where A is Asn, Asp or His, B is Val, Met or Phe, 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 G and Z togethar are also a covalent bond or -CO-(CH2)a-NH- where a is 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, -C6H5, -CH(OH)-CH3, , or -(CH2)b-T

(b being from 1 to 6 and T being hydrogen or OH, CH3O, CH3S, (CH3)2CH, C6H5, p-HO-C6H4, HS, H2N, HO-CO, H2N-CO, H2N-C(=NH)-NH) or M and Q together are a -(CH2)c-S-S-(CH2)d, -(CH2).-CO-NH-(CH2)r or -(CH2).-NH-CO-(CH2)8-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 theraof with physiologically toler-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-protective group, and M and Q are not con-nected 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, where G + Z together are a covalent bond or -CO-(CH2)a-NH-.

6. A peptide as claimed in claims 1 to 5 for use for con-trolling diseases.

7. The use of the peptides as claimed in claims 1 to 5 for controlling neoplastic diseases and autoimmune diseases as well as for control-ling and preventing infections, inflammations and transplant rejection reactions.

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