CA2005059A1 - Tnf peptides - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE: Peptides of the formula X-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

~o~o~9 O. Z . 0050/40387 NOVE~ TNF PEPl~IDES

The present invention relates to novel peptides derived from tumor necrosi~ factor (TNF), the preparation thereof and the usa thereof a~ drugs.

Car~well et al. (Proc. Natl. Acad. Sci. USA 72 (1975) 3666) reported that tha serum of endotoxin-treated animals which had previou~ly been infected with the Calmette-Guerin strain of Mycobacteria ~BCG) brought about hemorrhagic necro~is in various mouse tumors. ~his activity was a~cribed to tumor necrosis factor. TN~ also has a cytostatic or cytotoxic effect on a large number of tran3formed cell line~ in vitro, whereas normal human and animal cell lines are un~ffected (Lymphokine Reports ~ol.

2, pp 235-275, Academic Pres~, New York, 1981). Recently, the biochemical characterization and the gene for human TNF have been described ~Nature 312 (1984) 724, J. Biol.
Chem. 26~ (1985~ 2345, Nucl. Acids Res. 13 (1985) 6361~.

It is pos~ible to deduce from this data the following protein structure for ma~ure human TNFs V~ 3r~ -Dg~ ~r~9~9~y~k~VaL~k~sValVaLALU4~æro V~ ~VPlV~lP~U~Ly~eq~TSer GlnVrl~e~ysGlyG~$~yCy~sehrnT~sV~ fuTh~l~ohrIle S~gI~aV~l~an~sGh~7rLyaValA~ uS~U~IleLy~SerPrn GlyGlyValPi~luLy~yA~l~uI1dm~
~us~yt~nvalq~yIleIlea~

The TNP genes of ~attle, rabbits and mice have also been de~cribed ( Cold Spring Harbor Symp . Quant . Biol . 51 ( 1986 ) 597 ) .

~QO~;05i9 - 2 - O.z.0050/40387 Besides its cytotoxi~ properties, TNF is ~ne o~ the main substances involved in inflammatory reactions (Pharmac.
Res. 5 (1988) 129). Animal model8 have shown that TNF i~
involved in septic shock (Science 229 (1985) 869) and 5graft-ver~u~-host di~ea~e (J. Exp. Med. 166 (1987) 1280).

We have now found that peptide~ with a considerably lower mole~ular weight have beneficial propertie~.
The present invention relate3 to peptides of the formula I

X-A-Y I, where A is -Thr-Pro-Glu-Gly-Ala-, -Thr-Pro-Glu-Trp-Ala-, -Thr-Pro-Glu-Glu-Ala-, -Pro-Gly-Leu-Gln-Glu-Pro-, -Pro-Gly-Pro-Gln-Gly-Pro or -Pro-Gly-Le~-Gln-~ly-Pro-X is G-NH-CHN-CO-, G-NH-CHM-CO-W-, G-R-NH-CH~-CO- or G-R-NH-CHM-CO-W- and Y i~ -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 ic2 hydrogen or an amino-protective group, Z i~ OH or NH2 or a carboxyl-protective group, or G and Z together are al~o a coval~nt bond or -CO-(CH2),-NH- where a i8 from 1 to 12, R, U, V and W are peptide chains compo~ed of 1-4 natural-ly occurring ~-amino acid~ and N and Q ara hydrogens or one of the following -C~CH3)2, -CH(CH3)-C2H5, -C~H5, -CH(OH)-CH3, -CH2 ~ -CH ~ or -(CH2)b-T
H H
(with b bQing from 1 to 6 and T being hydrogen or OH, CH30, CH3S, (CH3)2CH, C~H5, p-HO-C~H~, HS, H2N, HO-CO, H2N-CO or H2N-C(=NH)-~H) or M and Q together are a -(C~2)c-s-s-(CH2) d- ~ - ( CH2 ) ,-CO-NH-(CH2)r~ or -(cH2).-NH-co-(cH2)~-NH-co-(cHz)~- bridge 2~

- 3 - O.Z.0050/40387 (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 as the salts thereof with phy~iologically toler-ated acids.

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

Particularly preferred physiologically tolerated acids are: hydrochloric acid, citric acid, tartaric acid, lactic acid, phosphoric acid, methane~ulfonic acid, acetic acid, formic acid, maleic acid, fumaric acid, malic acid, succinic acid, malonic acid, ~ulfuric acid, L-glutamic acid, L-aspartic acid, pyruvic acid, mucic acid, benzoic acid, glu~uronic acid, oxalic acid, ascor-bic acid and acetylglycine.

The novel pQptides can be open-chain (G = H, amino-protective group; Z = OH, NH2~ carboxyl-protective group, M and Q not connected ~ogether) and, in particular, have a disulfide bridge (G = H, amino-protec~ive group;
Z z OH, NH2, car~oxyl-protective group; N I Q - -(CH~)o~
S-S-(CH2)d-) or a side-chain bridge tG = H, a~ino-protec-tive group, Z ~ OH, N~2, carboxyl-protective group, M + Q
= - ( CH2 ) ~-NH-CO- ( CHz ) r~ or -(CH2).-NH-CO-(CH2)~-NH-CO-(CH2)s-) or be linked head-to-tail ~G + Z = covalent bond or -CO-(CH2),-NH-).

The novel compounds can be prepared by conventional methods of peptide chemistry.

Thu~, th~ peptide3 can be con~tructed sequentially from amino acids or by linking together ~uitable smaller peptide fragment~. In the sequential construction, the z~3050~9 _ 4 - o.Z.0050/40387 peptide chain is extended stepwise, by one amino acid each time, starting at the C terminus. In the ca~e of coupling of fragments it is possible to link together fragments of different lengths, these in turn being obtainable by sequential construction from amino acid~ or coupling of other fragments. The cyclic peptides are obtained, after synthesi~ of the open-chain peptides, by a cyclization reaction carried out in high dilution.

In the case both of sequential construction and of fragment coupling it i~ nece~sary for the building blocks to be linked by formation of an æmide linkage. Enzymatic and chemical methodY are ~uitable for thi~.

Chemical methods for forming amide linkages are deal~
with in detail by M~ller, Methoden der Organischen Chemie (Method~ of Organic Chemistry) Vol. XV/2, pp 1-364, Thieme Verlag, S~uttgart, 1974; Stewart, Young, Solid Phase Peptide Synthesi~, pp 31-34, 71-82, Pierce Chemical Company, Rockford, 1984; Bodanszky, Rlau~ner, Ondetti, Peptide Synthesis, pp 85-128, John Wiley & Sons, New York, 197~ and other ~tandard work~ of peptide chemistry.
Particularly preferred are the azide method, the symme-trical and mixed anhydride method, active e~ter~
generated in situ or preformed and the formation of amide link~ges using coupling reagents (activator~), in par-ticular dicyclohexylcarbodiimide (DCC), diisopropyl-carbodiimide ~DIC), l-ethoxycarbonyl-2-ethoxy-~,2-di-hydroquinoline(~EDQ),l-ethyl-3-(3-dimethyl~minopropyl)-carbodii~id~ hydrochlorid~ (EDCI), n propanephosphonic anhydride (PPA), N,N-bis(2-oxo-3-oxazolidinyl)zmido-phosphoryl chloride (BOP-Cl), diphenylphosphoryl azide [DPPA~, C~stro's reagent (BOP), O-benzotriazolyl-N,N,N',N'-tetra-methyluronium salt~ (HBTU), 2,5-diphenyl-2,3-dihydro-3-oxo-4-hydroxythioph~ne dioxide (Steglich's reagent; HOTDO) and l,l'-carbonyldiimidazole (CDI). The 5 O ~ 9 - 5 - O.Z.0050/40387 coupling reagents can be employed alone or in combination with additive~ such a~ N,N'-dimethyl-4-aminopyridine (DMAP), N-hydroxybenzotriazole (HOBt), N-hydroxybenzo-triazine (HOOBt), N-hydroxysuccinimide (HOSu) or 2-hydroxypyridine.

Whereas it is normally possible to di~pen~e with protec-tive groups in enzymatic peptide cynthesi~, for chemical synthesis it i~ nece~sary for there to ~e reversible protection of the reactive functional groups which are not involved in the formation of the amide linkage on the two reactant~. Three conventional protective group techniques ars preferred for chemical peptide synthe~es:
the benzyloxycarbonyl (Z), the t-butyloxycarbonyl (Boc~
and the 9-fluorenylmethyloxycarbonyl (Fmoc~ techniques.
In each case the protective group on the ~-amino group of the chain-extending building block i8 identified. The ~ide-chain protective groups on the trifunctional amino acid~ are chosen ~o that they are not necessarily elLmin-ated together with the ~-amino protective group. A
detailed re~iew of amino acid protective groups i~ given by N~ller, Methoden der Organischen Chemie Vol XV~l, pp 20-906, Thieme Verlag, Stuttgart, 1974.

The building block~ u~ed to construct the pQptidQ chain can be reacted in solution, in suspension or by a method ~imilar to that de~cribed by Merrifield in J. Amer. Chem.
Soc. 85 (1963) 2149. Particularly preferred method~ ar~
tho~e in which peptides are constructed sequentially or by fragm~nt coupling by use of the Z, Boc or Fmoc protec-tive group techniqu~r in which case the reaction takes place in solution, as well as those in which, similar to the Merrifield technique 3, one raactant i8 bound to an in~olubl~ polymeric support (also called resin herein-after). This typically entails the peptide b~ing con-~truct~d equentially on the polymoric support, b~ use of - 6 - O.Z.0050/40387 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 allows reagent~ and byproducts to be removed by filtration, and thus recrystallization of intermediates is superfluous.

The protected amino acid~ can he bonded to any ~uitable polymers which marely need to be insoluble in the sol-vents used and to have a stable physieal form which allows easy filtration. The polymer mu~t contain a functional group to which the first protected a~ino acid can be firmly linked by a covalent bond. A wide variety of polymers ia suitable for this purpose, for example cellulose, polyvinyl alcohol, polymethacrylate, ~ulfon-ated polystyrene, chloromethylated copolymer of styreneand divinylbenzene (Merrifield resin), 4-methylbenz-hydrylamine-re~in ~MBHA-re~in), phenylacetamidomethyl-resin (Pam-resin), p-benzyloxybenzyl alcohol-resin, benzhydrylamine-resin (~HA-resin), 4-hydroxymethyl-benzoyloxymethyl-re~in, the re~in used by Breipohl et ~1.
(Tetrahedron L~tt. 28 (1987) 565; from BACHEM), HYCRAN
resin (from ORP~ OE N) or SASRIN resin (from BACHEM).

Solvont~ ~uitable for peptide synthesis in ~olution are all those which are inert under the reaction conditions, in particular water, N,N-di~ethylformamide (DMF), dim0thyl sulfoxide (DMS0), acetonitrile, dichloromethane (DC~), 1,4-dioxane, te~rahydrofuran (THF), N-methyl-2-pyTrolidone (N~P) and mixtures of the ~aid solvents.
Peptide ~ynthesi~ on polymeric supports can b~ carried out in all inert organic solvents which dissolve the amino acid derivative~ u~ed; however, 801vent8 which also ha~e resin-sw~lling properties are preferred, such a~
DMF, DCM, ~MP, acetonitrile and DN~0, a~ wall as m~xture~
of the~e solvent~.

Z~:3050~9 - 7 _ O.Z.0050/403a7 After the peptide ha~ been synthe~ized it i cleaved off the polymeric support. The cleavage conditions for the variou~ type~ of resin~ are disclo~ed in the literature.
The cleavage reactions mo~t commonly use acid and palladium cataly~is, in particular cleavage in anhydrous liquid hydrogen fluoride, in anhydrouR trifluoromethane-sulfonic acid, in dilute or concentrated trifluoroacetic acid or palladium catalyzed cleavage in THF or THF-DCM
mixtures in the pre~ence of a weak base ~uch as morpho-line. ~he protective group~ may, depending on the choicethereof, be retained or likewise cleaved off under the cleavage condition~. Partial deprotection of the peptide may also be worthwhile if the intention i8 to carry out certain derivatization reaction3 or a cyclization.

Some of the novel peptide~ have good cytotoxic proper-ties. Some other~ of the peptides have high affinity for the cellular TNF raceptor without, however, having cytotoxic activity. They are therefore TNF antagonists.
They compete with natural TNF for binding to the cellular TN~ receptor and thu~ suppress the TNF effect. The novel peptide are valuable drugs which can be employed for treating neoplastic diseases and autoi une diseases as well a~ for controlling and preventing infection~, inflammation~ and transplant reJection reactions. Simple experiment~ can be u ed to elucidate the mode of action of the individual peptides. The cytotoxicity of the peptide i~ determined by incubating a TWF-~ensiti~e cell line in the presence of the peptide. In a second experi-mental approach, the cell line i8 incubated with the relevant peptide in the presence of a lethal amount of TWF. It is possible in this way to detect the TNF-antagonistic effect. In addition, the affinity of the peptide for the cellular TNF rsceptor i~ determined in an in vitro bindin~ e~periment.

Z~U50~9 - 8 - O.Z.0050/40387 The following test systems were used to characterize the agonistic and antagonistic effects of the novel peptide~:

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

I. Cytotoxicity te~t The agonistic effects of the novel peptides are assessed on the basis of their cytotoxic effect on TNF-sensitive cells (s.g. L929, MCF-7, A204, U937).
The test with L929 and MCF-7 was carried out a~
follows:

1. 100 ~1 of cultura medium containing 3 to 5 x 103 freshly trypsinized, exponentially growing, L92g cells ~mouse) or MCF-7 cells (human) were pipetted into the wells of a 96-well flat-bottem culture plate. Th~ plate wa~ incubated at 37-C
overnight. The air in th~ incubator ~as saturated with water vapor and contained 5% Cl)2 by volume.

The L929 culture medium contained 500 ml of lx Earle'~ M~M (Boehringer Nannheim), 50 ml of heat-inactivated (56C, 30 min) fet~l cAlf serum (FCS), 50 ml of L-glut~min~ (200 mM), 5 ml of lOOx non-e~sential amino acids, 3 ml of lM HEP~S
buffer pH 7.2,and 50 ml of gentamicin (50 mg/ml).

The MCY-7 culture medium contained 500 ml of lx Dulbecco'~ MæM (Boehringer Na~nhe~m), 100 ml of heat-inhctivated (56C, 30 min) FCS, 5 ml of L-glutamin~ and 5 ml of lOOx non-essential amino acids.

z~o~o~9 - 9 - O.Z.0050/40387 2. The next day 100 ~1 of the peptide solution to be te~ted were added to the cell cultures and sub~ected to erial 2-fold dilution. In addition, some cell controls (i.e. cell culture~ not treated with peptide dilution) and some rhu-TNF
controls (i.e. cell culture~ treated with recom-binant human TNF) were also made up. The culture plate was incubated at 37 DC in an atmosphere of air saturated with water vapor and containing 5 C02 by volume for 48 h.

3. The percentage of 3urviving cells in the culture~
treated with peptide dilution was determined by staining with crystal violet. For this purpose, the liquid wa~ removed from the well~ of the te3t plate by tapping it. 50 ~1 of cry~tal violst solution were pipetted into each well.

The co~position of th~ cry~tal violet solution wa~ as follow3 5 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 cry~tal violet solution wa~ left in the wells for 20 min and then likffwise removed by tapping.
The plates wore then wa~hed 5 time~ by immerslon in w~ter in order to re~ove dye not bound to the cell~. The dye bound to the cells wa~ e~tracted by adding 100 ~1 of re~g~nt ~olution (S0~ etha-nol, 0.1% glacial acetic acid, 49.9% water) to each well.

2C3050~
- 10 - O.Z.0050~40387 4. The plates were shaken for 5 min to obtain a solution of uniform color in each well. The surviving cell3 were determined by measuring the extinction at 540 nm of the colored solution in the individual well~.

5. SubYequently, by relating to the cell control, the 50% cytotoxicity value wa~ defined, and the reciprocal of the sample dilution which re~ulted in 50~ cytotoxicity was calculated a~ the cyto-toxic activity of the te~t ~ample.

II. Cytotoxicity antagonism test The antagoni3tic effect of the peptide~ was assessed on the ba~is of their property of antagonizing the cytotoxic effect of rhu-TNF on TNF-senqitive Cell8 (e.g. ~929, MCF-7, A204, U937). Tha cytotoxicity antagonism te~t with L929 and MCF-7 cells wa~
carried out as follow~:
1. 100 ~l of culture mediu~ containing 3 to 5 x 103 fre~hly trypsinized, exponentially growing, L929 calls (mou~e) or MCF-7 cell~ (human) were pipetted into the wells of a 96-well flat-bottom culture plate. The plate wa~ incubat~d at 37-C
overnight. The air in the incubator wa~ saturated with water vapor and contained 5% CO2 by volume.

The ~92~ cultura medium cont~ined 500 ml of lx Earle ~ 8 ME~ (Boehringer Mannhe~m), 50 ml of heat-inactivated ~56-C, 30 min) FCS, 5 ml of L-gluta-minQ (200 mM), 5 ml of lOOx non-e~antial amino acid~, 3 ml of lN H~PES buffer p~ 7.2, and 500 ~l of gentamicin (50 mg/ml).

The MCF-7 cultur~ medium contained 500 ml of lx Dulbecco'~ MEM (Boehringer Mannhei~), 100 ml of ~ O.Z.0~50/40387 heat-inactivated (56C, 30 min) FCS, S ml of L-glutamine (200 mM) and 5 ml of lOOx non-essen-tial amino acids.

2. The next day 100 ~1 of the peptide 301uticn to be te~ted were added to the cell cultures and ~ubjected 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 the~e cell culture~. In addition, ~ome cell controls (i.e. cell cultures not treated with peptide solution or with rhu-TNF solution) and ~ome rhu-TWF controls (= cell cultures trested only with rhu-TNF ~olution) were also made up. The culture plate was then incubated at 37C in an atmosphere of a~r saturated with water vapor and containing 5% CO2 by volume for 48 h.

3. The percentage of ~urviving cells in the cultures treated with ~ubstance solution wa~ determined by staining with crystal violat. For thi~ purpo~e, the liquid wa~ removed fro~ the w~118 of the test plate by tapping it. 50 ~1 of cry~tal violet solution w~re pipetted into each wQll.

The cry~tal violet solution had the compo~ition ~pecified in I.3 The cry#tal violet ~olution wa~ left in the well~
for 20 min and then lik~wise removed by tapping.
The plate~ wers then w~shed 5 ti~es by i~mersion in water in order to rQmove dye not bound to the cell~. The dye bound to the cell~ was axtracted by adding 100 ~1 of reagen~ solution (50% etha-nol, 0.1% glacial acetic acid, 49.9~ water) to Z~3050~i9 - 12 - O.Z.0050/40387 each well.

4. The plates were shaken for 5 min to obtain a solution of uniform color in each well. The surviving cell~ were determined by measuring the S extinction at 540 nm of the co~ored ~olution in the individual wells.

5. Subsequently, by relating to the cell control and the rhu-TNF control, the 50% antagoni~m value wa~
defined, and the sample concentration which re~ulted in 50% antagonism of rhu-TNF cytotox-icity at the rhu-TNF concentration used wa8 calculated a~ antagonistic activLty of the 8ample tested.

III. Competitive receptor-binding test Both the agonistic and antagoni~tic effects of peptides are conditional on the latter binding to the T~F receptor. This means that peptides with an agonistic or antagoni~tic effect compete with rhu-TNF for binding to the TNF receptor on TNF-~ensitive indicator cell~ te.g. U937). The competL-tive receptor-binding te~t was carried out a~
followss 1. 100 ~1 of medium containing variou~ concentra-tion~ of thQ peptide to be te~ted and of rhu-TNF
(- control) were pipetted into the reaction vessel~. The medium comprised 500 ml of PBS
(Boehringer Mannheim) containing 10 ml of heat-inact~vated (56C, 30 min) FCS and 100 mg of Qodium azide.

2. Subsequently, 100 ~1 of ~edium containing 1 ng of l2~I-labeled rhu-TNF (Bolton lactoperox~dase zoo~osg - 13 - O.Z.0050/40387 method) were placed in the reaction ~essels and mixed. The non-specific binding (NSB) was deter-mined by mixing in the reaction vegsel~ the l25I-labeled rhu-TNF (1 ~g of l25I-rhu-TNF in 100 ~l of medium) with a 200-fold exces~ of unlabeled rhu-TNF (200 ng of rhu-TNF in 100 ~l of medium).

3. Then 100 ~l of medium containing 2 x 10~ U937 cQlls (human) were pipetted into the reaction vessel~ and mixed. The reaction vessel~ (te~t volume 300 ~l) were incubated at 0~C for 90 min.
The reaction mixture~ were remixed after 45 min.

4. After the incubation the Cell8 were centrifuged at 1800 rpm and 4C for 5 min, washed 3 times with medium and tran~ferred quantitatively into counting vials, and the cell-bound radioactivity was determined in a Clini gamma counter 1272 (LRB Wallac).

5. After the measurement~ had been corrected for the non-~pecific binding, the 50~ compQtition value wa~ defined by relat~on to the overall binding, and the ~ample concentration which led to 50%
co~petition of ~5I-rhu-TNF bindinq at th0 ~I-rhu-~NF concentration used W83 calculated as the competitive activity of the sample te~ted.

~he ~xamples which follow are intendod to explain the invention in more detail. The proteinogenou~ ~m~no acids are abbroviated in the Examples using the conventional three-letter code. Other meaning~ ares AC = acetic acLd, Hcy = homocysteLne, Orn = ornithine, Dap = 2,3 dia~inopropionic ac~d.

z~osos9 - 14 - O.Z.0050/~0387 A. General procedures I. The peptide~ claimed in claim 1 were synthe~ized using standard method3 of solid-phase peptide synthesi~ in a completely automatic model 430A
peptide synthesizer from APPLIED BIOSYSTENS. The apparatus u~es different synthesis cycle~ for the Boc and Fmoc protective group techniques.
.

a) Synthesi~ cycle for the Boc protectivs group technique 1. 30% trifluon~ic acid in DCM 1 x 3 min 2. 50% trifluanoYIkic acid in DaM 1 x 17 min 3. DC~w3~hing 5 x 1 min 4. 5% dlLY~elFylethyla~ne in DCM 1 x 1 min 5. 5% dl1u~YpylathyLamine in ~P 1 x 1 min 6. NMP wRshlng ~ 5 x 1 mLn 7. ~dition of ~n~tivated prvuY1ed amino acid (astiva~ion ~y 1 ~;va~ of DOC
and 1 equi~alent of HaBt in ~MP/DCM);
p ~ coupling (lst part) l x 30 min 8. Additlon of n~so to the neY~cn m~ e until it o~ ns 20% DMSO by ~lume 9. Pq~b coupling (2nd part~ 1 x 16 min 10.A~itiQn of 3.8 oy~iva~ of ~iion-ll.Pq~i~b c3upling (3rd part) 1 x 7 m~
12.DCNwa~ng 3 x 1 min 13.If n~ is ~xxwp~, rq~ition of co~inq (r~h~n bo 5.) 14. 10% ~ ~nr~h~de, 5% dLL~¢cQyl-ethyLa~ne in DCN 1 x 2 min 15. 10% ~o~i~ a ~ idb in DC~ 1 x 4 min 16.DCN wa~ng 4 x 1 min 17 ~h~n bo 1.

ZOIQ505~
- 15 - O . Z . 0050/40387 b) Synthe~ le for the E~c protec~ive gr~up tec~iql~!e 1. NMP washing . 1 x 1 min 2. 20% piperidine in NMP 1 x 4 min 3. 20% piperidine in I~P 1 x 16 min 4. ~e wa~ g 5 x 1 min 5. ~ition of E~ tivated ~1 amino a~id (a~tivaticn ~y 1 eq!livalent of OCC
and 1 eq!livalent of ~t in NMP/DCM);
pepti~3e ~lir~g 1 x 61 min 6. ~le wa~hi~ 3 x 1 min 7. }f reacti~ ;~ plete, r~iti~ of ca~ling (~et~ to 5. ~
8. 109~ a~G ar~id~ in NMP 1 x 8 min 9. NMP wa~hing 3 x 1 min 10. ~n ~ 2.

II. Working up of peptide-resins obtained a8 in Ia The peptide-res$n obtained as in Ia wa~ dried under reduced pres~ure and transferred into a reaction ve3sel of a Teflon HF apparatus ( from PENINSULA) .
Addition of a ~cavenger, preferably anisole (1 ml/g of resin), and of a thiol, in the ca~e of tryptophan-contsining peptides, to remove the indole for~oyl group, pre~erably ethanedithiol ~O.S ml/g of resin), was followad by condensation in of hydrogen fluoride (10 ml/g of resin) while cooling with liquid N2. The mixture was allowed to warm to O'C, and was ~tirred at thi~ temperature for 45 min. The hydrogen fluor-ide wa~ then stripped off under reduced pre~sure and the re~idue wa~ washed with ethyl acetate in order to remove remaining ~cavenger. The pepti de wa3 extracted with 30% tr~ngth acetic acid and filterad, and the filtrate was freeze-dried.

~o prepare peptide hydrazides, the peptide-resin Pam- or ~errifield resinJ was suspended in DME

2QOSO~i9 - 16 - O.Z.0050/40387 (15 ml/g of re~in), hydrazine hydrate (20 equiva-lents) was added, and the mixture wa~ stirred at room temperature for 2 days. To work up, the resin wa~ filtered off and the filtrate wa~ evaporated to dryness. The residue was cry~tallized from DMF/Et20 or MeOH/Et2O.

III. Working up of the peptide-re~ins obtained a~ in Ib The peptide-resin obtained as in Ib wa~ dried under reduced pressure and subsequently sub~ected to one of the following cleavage procedure~, depending on the amino acid composition (Wade, Tregear, Howard Florey Fmoc-Workshop Manual, Melbourne 1985).

Peptide containing Cleavage conditions ~rg(Mtr) Met ~rp TFA Scavenger Reaction TLm~

no no no 95% 5% H2O 1.5 h yes no no 95% 5% thioanisole ~ 3 h no ye~ no 95% 5~ ethyl methyl 1.5 h ~ulfide no no yes 95% 5% ethan~dithiol/1.5 h anisole (ls3) no yes yes 95% 5% ethanedithiol/ 1.5 h ani~ole/ethyl methyl sulfide (1:3sl) ye~ ye~ ye~ 93% 7~ ethanedithiol/ ~ 3 h anisol~/ethyl methyl sulfide (ls3s3) -The ~u~pen~ion of the peptide-re~in in th~ suitable TFA mixtur~ wa~ 3tirred at roo~ temperature for the 2~05059 - 17 - O.Z.0050/40387 stated time and then the re~in wa~ filtered off and washed with TF~ and with DCM. The filtrate and the wa~hing~ were exten~ively concentrated, and the peptide was precipitated by addition of diethyl ether. The mixture wa~ cooled in an ica bath, and the precipitate was filtered off, taken up in 30%
acetic acid and freeze-dried.

IV. Purification and characterization of the peptides Purification wa~ by gel chromatography (SEPHADEX~
G-10, G-15/10% HOAc; SEPHADEX LH20/MeOH) and ~ub 3equent medium pres~ure chromatography lstationary pha~es HD-SIL C-18, 20-45 ~, lo0A; mobile pha~e:
gradient with A = 0.1% TFA/MeOH, B = 0.1% TFA/H2O).

The purity of the final products was determined by analytical HPLC (stationary phaQes 100 x 2.1 mm VYDAC C-18, 5 ~, 300 ~; mobile phase = CH3CN/H20 gradient buffered with 0.1~ TFA, 40C). Charac-terization was by means of amino acid analysis and fast atom bo~bardment mass spectrometry.

~. Specific procedure3 EXAMPL~ 1 Ac-Ser-Pro-Thr-Gln-Arg-Glu-Thr-Pro-GlurGly-Ala-Glu-Ala-Ly8 -Pro-Trp-TYr-NH2 0.98 g of Boc-Tyr (Br-Z~ MBHA-re~in (~ubstitution 0.51 mmol/g), corresponding to a b~tch size of 0.5 mmol, wa~
reacted as in AIa with 2 mmol each of Boc-Trp(CHO)-OH Boc-Gly-OH Boc-Arg(To~)-OH
Boc-Pro-OH Boc-Glu(OChx~-O~ Boc-Gln-O~
Boc-Ly~(Cl-Z)-OH Boc-Pro-OH Eoc-Thr(Bzl)-OH

- 18 - O.Z.0050/40387 Boc-Ala-OH Boc-Thr(Bzl)-OH Boc-Pro-OH
Boc-Glu~OChx)-OH Boc-Glu(OCHx)-OH Boc-Ser(Bzl)-OH
Boc-Ala-OH

After the synthesi~ was complete, the N ~erminus wa~
acetylated (~tep~ 1-6 and 14-16 as in AIa). The peptide-resin wa~ dried under reduced pres~ure; the yield was 2.2.g.
1.1 g of the resin obtained in this way were ~ub~ected to HF cleavage as in AII. The crude product (411 g) was purified by gel filtration (SEPHADEX0 G-10) and medium pressure chromatography (cf. AIV; 50-65% A; 0.25~ min1).
242 mg of pure product were obtained.

H-Met-Val-Tyr-Pro-Gly-Leu-Gln-Glu-Pro-Trp-Leu-OH

0.47 g of Fmoc-Leu-p-alkoxybenzyl alcohol-re~in (~ubstit-ution 0.53 mmol/g), corre~ponding to a batch ~ize of 9.25 m~ol, wa~ reacted a~ in AIb with 1 m~ol each of Fmoc-Trp-OH Fmoc-Gly-OH
Fmoc-Pro-OH Fmoc-Pro-OH
Fmoc-Glu(OtBu)-OH Fmoc-Tyr(t~u)-OH
F~oc-Gln-O~ Fmoc-Val-OH
Fmoc-Leu-OH Fmoc-Met-OH

After the ~ynthesis was completa, the peptide-re~in underwent N-terminal deprotection (step~ 2-4 a~ in AIb).
The resulting peptide-reain wa~ dried under reduced pressure; the yield was 0.72 g.

~he crude peptide (251 mg) obtained after TFA cloa~age as in AIII wa~ purified by gel filtration (S~PHADEX~ G - 10) and medium pre~sure chromhtography (cf. AIV; 60- S% ~;
0.25% min~l). 193 mg of pure produc~ were obtained.

2~305059 - 19 - O.Z.0050~40387 The following can be prepared in a ~imilar manner to Examples 1 and 2:

3. H-Ser-Prr-Thr-Gln-Arg-Glu-Thr-Pro-Glu-Gly-Ala-Glu-Ala-Ly~- ~-Trp-Tyr-OH 4. H-Ser-Pro-Tyr-Gln-Arg-Glu-Thr-Pro-Glu-Gly-Ala-Glu-Ala-Lys-Pro-~rp-ryr-OH
5. Ac-Ser-Pro-~yr-Gln-Arg-Glu-Thr-Pro-Glu-Gly-Ala-Glu-- Ala-Ly~-Pro-Trp-Tyr-NH2 6. Ac-Met-Val-Tyr-Pro-Gly-Leu-Gln-Glu-Pro-Trp-Leu-NH2 Ac-Hcy-Gln-Arg-Glu-Thr-Pro-Glu-Gly-Ala-Glu-Ala-Ly~-Pro-~y-NH2 150.98 g of Boc-HcytpMB)-MBHA-re~in (substitution 0.51 mmol/~), corresponding to a b~tch siz~ of 0.5 mmol, wa~
reacted a3 in AIa with 2 mmol each of Boc-Pro-OH Boc-Pro-O~
Boc-Ly8(Cl-Z)-OH Boc-Thr(Bzl)-OH
Eoc-AlA-OH Boc-Glu(OChx)-OH
Boc-Glu-(OChx)-O~ Boc-Arg(Tos)-OH
Boc-Ala~O~ Boc Gl~-OH
Boc-Gly-OH Boc-Hcy(p~B)-OH
Boc-Glu(OChx)-OH

25 After the synthe~is was complete, the N terminus w~
acetylated ~step~ 1-6 and 14-16 a~ in AIa). The peptide-re~in wa8 dried under reduced pre~ure; ~he yield was 1.55 g.

0 5 ~ ~ 9 - 20 - O.Z.0050/40387 0.78 g of the re~in obtained in this way was sub~ected to HF cleavage as in AII. The freeze-dried cruda product wa~
taken up in 2 1 of 0.1% strength acetic acid, and the pH
was then ad~usted to 8.4 with aqueou~ ammonia. Under an argon atmosphere, 0.01 N ~31Fe(CN)6] 301ution was slowly added dropwise until the yellowish-gre2n color persisted for at leàst 15 min. The mixture was then stirred for 1 h and then acidified to pH 4.5 with glacial acetic acid, and 15 ml of an aqueous su~pen~ion of an anion exchanger ~BIORAD 3 x 4A, chloride form) were added. After 30 mln, the ion exchanger resin was filtered off, and the filt-rate was concentrated to 100 ml in a rotary evaporator and ~ubsequently freeze-dried.

All the solvents used had previou~ly been ~aturated with nitrogen in order to prevent any oxidation of the free cysteina re~idue~.

The crude product was purified by gel chromatography (SEPHAD~X G-15) and medium pres~ure chromatography (cf.
AIV; 40-60% A; 0.25% min1). 58 mg of pure product were obtained.

The following can b3 prepared in a ~imilar manner to Example 7 (Pam-resin wa~ used to ~ynthe~ize the peptide acid~)s .
8. H- ~ s-Gln-Arg-Glu-Thr-Pro-Glu-Gly-Ala-Glu-Ala-Lys-~ ' Pro-Cys-OH
9. Ac-Cys-Gln-Arg-Glu-Thr-Pro-Glu-Gly-Ala-Glu-Ala-Lys-Pro-Cys-~H2 10. ~-Hcy-Gln-Arg-Glu-Thr-Pro-Glu-Gly-Als-Glu-Ala-Ly~-I
Pro-Cys-OH

~(~05059 - 21 - O.Z.0050/40387 11. Ac-Hcy-Gln-Arg-Glu-Thr-Pro-Glu-Gly-Ala-Glu-Ala-Lys-Pro-cys-NH2 s 12. H-Cy~-Gln-Arg-Glu-Thr-Pro-Glu-Gly-Ala-Glu-Ala-~ys-I
Pro-Hcy-OH
-13. Ac-C~s-Gln-Arg-Glu-Thr-Pro-Glu-Gly-Ala-Glu-Ala-Lys-pro-Hcy-NH2 14. H-Hc ~ ln-Arg-&lu-Thr-Pro-Glu-Gly-Ala-Glu-Ala-Ly~-Pro-Hcy-OH

15. Ac-C~-Pro-Gly-Leu-Gln-Glu-Pro-Cys-NH2 16. H-Cys-Pro-Gly-Leu-Gln-Pro-Cys-OH
i 17. H-Hcy-Pro-Gly-Leu-Gln-Glu-Pro-Cys-OH

18. Ac-HLy-Pro-Gly-Leu-Gln-Glu-Pro-Cy8-NH2 r 19. H-Cys-Pro-Gly-Leu-Gln-Glu-Pro-Hcy-OH

20. H-Cy~-Thr-Pro-Glu-Gly-Ala-Glu-Ala-Cys -NH2 21. as-Cy~-Thr-Pro-Glu-Gly-Ala-Glu-Ala-Cys-NH2 22. H-Cys-Pro-Glu-Gly-Ala-Glu-Cys-NH2 ,.

23. Ac-Cys-Pro-Glu-Gly-Ala-Glu-Cy~-NH2 _ i 24. H-Cy~-Pro-Glu-Gly-Ala-Cys-NH2 25. A~-Cy~-Pro-Glu-Gly-Ala-Cy~ -NH2 - 22 - O.Z.0050/40387 26. Ac-Cy~-Pro-Gly-Leu-Gln-Glu-Pro-Hcy-NHz 27. H-Hcy-Pro-Gly-Leu-Gln-Glu-Pro-Hcy-OH
r 28. Ac-H~y-Pro-Gly-Leu-Gln-Glu-Pro-Hcy-NH2 29. Ac-Lys-Ser-Pro-Cys-Gln-Arg-Glu-Thr-Pro-Glu-Gly-Ala-Glu-Ala-hys-Pro-Cy~-Tyr-Glu-Pro-N~I2 30. Ac-Ile-Ly~-Ser-Pro-Hcy-Gln-Arg-Glu-Thr-Pro-Glu-Gly-Glu-Ala-Ly~-Pro-Hly-Tyr-Glu-Pro-NX2 1 ~

31. A~-Ly~-Net-Val-Hcy-Pro-Gly-Leu-Gln-Glu-Pro-H y-Leu-Hi8-Ser-~2 32. Ac-Lys-Ser-Hcy-Ser-Gln-Arg-Glu-Thr-Pro-Glu-Gly-Ala-Glu-Ala-Lys-Pro-Trp-Hcy-Glu-Pro-NH2 33. Ac-Il~-Lys-Hcy-Pro-Ser-Gln-Arg-Glu-Thr-Pro-Glu-Gly-~ I
Al~-Glu-Ala-Ly~-Pro-Trp-Tyr-Hcy-Pro-Ile-NH2 Ac-L~-Me~-Val-Tyr-Pro-Gly-Lsu-Gln-Glu-Pro-~u-NH2 1 g of rs~in described by Breipohl et al. (from BACHEN), corresponding to a b~tch size of 0.5 mmol/ w~ reacted a~
in AIb w$th 2 m~ol ~ch of Fmoc-Glu(OtBu)-OH Fmoc-Leu-OH Fmoc-Val-OH
Fmoc-Pro-OH Fmoc Gly-O~ Fmoc-M~t-O~

~æ~30rJoss - 23 - O . Z . 0050/40387 E moc -Glu ( OBz 1 ) -OH E moc -Pro-OH Fmoc -Lys ( Boc ) -OH
Fmoc-Gln-OH Fmoc-Tyr~tBu)-oH

After the synthesis was complete, the N terminus was acetylated (step~ 2-4 and 8-9 as in AIb). The peptide-resin wa3 dried under reduced pres3ure; yield 1.86 g, The crude product t615 mg) obtained after TFA cleavage as in AIII wa di~solved in 500 ml of dega~sed DNF. 0.24 ml of NEt3 and then, at -25C, 0.24 ml of diphenylphosphoryl azide were added and the mixture wa~ ~tirred at -25C for 2 h. It was ~ubsequently stored at -20C for 2 day~, at 4C for 2 days and at room temperature for 2 days. It wa~
then evaporated to dryne~s, and the crude peptide was purified by gel chromatography (SEPHADEX~ LH 20). The isolated monomer (122 mg~ was deprotected with HP a~ in AII and puri~ied by medium pressure chromatography (cf.
AIV, 45-60~ A; 0.25% minl). 83 mg of pure product were obtained.

EXAMPL~ 35 H-Ser-Ser-Gln-~y~-M~-Val-Tyr-Pro-Gly-Leu-Gln-Glu-Pro-Glu-Leu-Kis-Ser-OH

2.63 ~ of Boc-Ser(Bzl)-~errifield re~in (substitution about 0.38 ~.,ol/g), corresponding to ~ batch ~ize of 1 mmol, wore reacted a~ in AIb with 4 mmol each of Boc-His(ZJ-OH Fmoc-Leu-OH Fmoc-Met-OH
Boc-Leu-OH Fmoc-Gly-OH Fmoc-Ly~-(Boc)-OH
Fmoc-Glu(OtBu)-OH Fmoc-Pro-OH Fmoc-Gln-OH
Fmoc-Pro-O~ Fmoc-Tyr(tBu)-O~ Fmoc-Ser(Bzl)-O~
Fmoc-Glu(OBzl)-OH Fmoc-V~l-OH Fmoc-Ser(B21)-OH
Fmoc-Gln-OH

2~30505~

- 24 - O.Z.005~/403~7 Subsequently the t-butyl and Boc protective group~ were cleaved off (steps 1-6 a~ in AIa). The cyclization on the resin took place in NMP with the addition of 1.77 g of ~OP and 1.74 ml of diisopropylethylamine (16 h). The S peptide-resin underwent N-terminal deprotection (step~ 2-4 as in AIb) and drying under reduced pre3sure. The yield wa3 3.51 g. The crude product obtained after HF cleavage as in AII wa~ purified by gel filtration (SEPHADBX~ G-25) and medium pr~ssure chromatography twic~ ~cf. AIV; 40 to 60g A; 0.25% min~l). 23 mg of pure product were obtained.

The following can be pr~pared in a sLmilar manner to Examples 34 and 35s 36. Ac-Lys-Met-Val-Tyr-Pro-Gly-Leu-Gln-Glu-Pro-Glu-NH~
37. Ac-Lys-Met-Val-Tyr-Pro-Gly-Leu-Gln-Glu-Pro-Glu-OH
I
38. Ac-Lys-Ser-Pro-Thr-Gln-Arg-Glu-Thr-Pro-Glu-Gly-Ala-Glu-Ala-Ly~-Pro-Trp-Tyr-~ p-NH2 39. A~-Lys-Thr-Pro-Glu-Gly-Ala-Asp-NH2 40. H-Lys-Thr-Pro-Glu-Gly-Ala-~sp-OH
41. As-Glu-Thr-Pro-Glu-Gly-Ala-Ly8-OH

42. Ac-Ly~-Ser-Pro-Tys-Gln-Arg-Glu-Thr-Pro-Glu-Gly-Ala-Glu-Ala-Lys-Pro-Trp-Tyr-Glu-N~2 r- --43. Ac-Orn-Ser-Pro-Tyr-Gln-Arg-Glu-Thr-Pro-Glu-Gly-Ala-Glu-Ala-Ly~-Pro-Trp-l!yr-A8p-NE~2 Z ~ ~ 5 0 S9 - 25 - O.Z.0050/40387 44. Ac-Ly~-Pro-Thr-Gln-Arg-Glu-Thr-Pro-Glu-Gly-Ala-Glu-Ala-Lys-Pro-Trp-Glu-NH2 1 - .
45. Ac-Glu-~hr-Gln-Arg-Glu-Thr-Pro-Glu-Gly-Ala-Glu-Ala-Ly8-Pro-Ly8-NH2 46. Ac-A3p-Gln-Arg-Glu-Thr-Pro-Glu-Gly-Ala-Glu-Ala-Ly~-Pro-O~n-NH2 47. ~-Ly3-Pro-Gly-Leu-Gln-Glu-Pro-Glu-NH2 , 48. Ac-Lys-Pro-Gly-Leu-Gln-Glu-Pro-Glu-OH

49. Ac-Ser-Ser-Gln-L~s-~et-Val-Tyr-Pro-Gly-Leu-Gln-Glu-Pro-Glu-Leu-His-Ser-NH2 ~Gly-Leu-Gln-Glu-Pro-Trp-Leu-Tyr-Prol 1.11 g of F~oc-Pro-p-alkoxybenzyl alcohol-resin (substit-ution 0.45 mmol/g), corresponding to a batch ~ize of 0.5 mmol, were r~acted as in AIb with 2 mmol each of Fmoc-Tyr(tBu)-OH Fmoc-Glu(QBzl)-OH
Fmoc-Leu-OH Fmoc-Gln-O~
Fmoc-Trp-OH Fmoc-~eu-OH
Fmoc-Pro-OH Fmoc-Gly-O~

After the synthQsis was complete, the peptide-re~in 200~05g - 26 - O.Z.0050/40387 underwent N-terminal deprotection (step~ 2-4 a~ in AIb), and subsequent drying undar reduced pre~sure. The yield was 1.55 g.

The cruds peptide (486 mg) obtained after TFA cleavage as in AIII wa8 dissolved in 500 ml of de~aosed DMF. 210 mg of NàHCO3 and then, at - 25C, 0.24 ml of diphenylphos-phoryl azide were added, and the mixture waq ~tirred at - 25C for 2 hours and at room tempsrature for 2 days. It was then evaporated to dryness, and the crude peptide was purified by gel chromatography (SEPHADFX~ LH 20). The isolated monomer (73 mg) was deprotected with HF a~ in AII and purified by med~um presaure chromatography (cf.
AIV; 60-70% A; 0.25~ minl). 32 mg of pur~ product were obtained.

The following can be prepared in a ~imilar manner to Example 50:

51. ~Pyr-~ln-Arq-Glu-Thr-Pro-Glu-GlY-Ala-Glu-Ala-Lys-Pro -Trp~

52.rLyL_ M~t-Val-Tyr-Pro-GlY-Leu-Gln-Glu-Pro-Glu~

53.~Yr-Pro-~ly-Leu-Gln-Glu-Pro-Trp_,

Claims (8)

1. A peptide of the formula I, X-A-Y I, where A is , , , or X is , , or 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)4-NH- where a is from 1 to 12, R, U, V and W are peptide chains composed of 1-4 natural-ly 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
(with b being from 1 to 6 and T being hydrogen or OH, CH3Oo, CH3S, (CH3)2CH, C5H5, p-HO-C?H4, HS, H2N, HO-CO, H2N-CO or H2N-C(=NH)-NH) or M and Q together are a -(CH2)c-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 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 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-(CHz)f- or -(CH2)?-NH CO
(CH2)5-NH-CO-(CH2)f.

5. A peptide as claimed in claim 1, where G + Z toge-ther 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 reaction reactions.

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