CA2005058A1 - Tnf peptides - Google Patents

Abstract

Abstract of the Disclosure: Peptides of the formula X-A-Gly-Asp-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

2Q0505~

O.Z. 0050/40388 ; NOVEL TNF PEPTIDES

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

Carswell et al. (Proc. Natl. Acad. Sci. USA 72 (1975) 3666) reported that the serum of endotoxin-trea~ed animals which had previously been infected with the Calm~tte-Guerin strain of Mycobacteria (BCG) brought about hemorrha~ic necrosis in variou~ mouse tumors. This activity was ascribed to tumor necrosi3 factor. TNF also has a cyto~tatic or cytotoxic effect on a large number of transformed cell lines in ~itro, wherea~ normal human and animal cell lines are unaffected (Lymphokine Reports Vol.

2, pp 235-275, Academic Pre~s, New York, 1981). Recently, the biochemical characterization and the gene for human TNF have been described ~Nature 312 ~1984) 724, J. Biol.
Chem. 260 (1985) 2345, Nucl. Acid~ Res. 13 (1985) 6361).

It is po~sible to deduce from this data the following protein structure for mature humsn TNFs V~IOn~r~ n~eePr3i~spLy~VaL~k~ValV~L~aA~

VA~
GlnV~L~3~b-Ly2GlyE~yCy~sSerTh~V~ v~h~e~lle ~ gI~aVal;~1~rG~tnrLysV~IA~ dU~IleLya~ o Cy G~Yb~l~ uGlyA~ ~ a~y ~xnrpry~uProIl~ieu ayV~l~h~l~yA~r~uI~e.~r~
~yruaOuFPh3L~kau~ yGanVal~y~Y~ylleI~e~eu The TNF gene~ of c~ttle, rabbit~ and mice have al~o been described (Cold Spring ~arbor Symp. Quant. Biol. 51 tl986) 597).

- 2 - O.Z. 0050/40388 Be~ides it~ cytotoxic properties, TNF is one of the main ~ubstances involved in inflammatory reactions (Pharmac.
Re~. 5 (1988) 129). Animal model~ have 3hown that TNF i~
involved in septic shock (Science 229 (1985) 869) and S graft-versus-host disea~e (J. Exp. Med. 166 (1987) 1280~.

We have now found that peptides with a con~iderably lower molecular weight have beneficial properties.

~he pre~ant invention relate~ to peptides of the formula I
X-A-Gly-A~p-Y
where A is Lys, Gln or Arg, X iB G-NH-CH~-CO-, G-NH-CHX-CO-W-, G-R-NH-CH~-CO- or G-R-NH-CHM-CO-W- and 15 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-protectiv~ group, Z is ON or NH2 or a carboxyl-protective group or G and Z together are also a covalent bond or -CO-(CH2),-NH-, where a i~ 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)-C2H5, -C~H5, -CH(OH)-CH3, -CH 2~ ~ or - ( CH2 ) b-T
H H
~with b being from 1 ~o 6 and T baing hydrogen or OH, CH30, CH3S, (CH3)2CH, C~H~, p-HO-C~H~, HS, H2N, HO-CO, H2~-CO, H2N-Ct-NH)-NH) or M and Q together are a -(CH2Jo-S-S-(CH23d, -(CH2).-CO-NH-(cH2)r or -(CH2).-NH-co-(cH2)~-N~-co-(cHz) brldge twith c and d ~eing from 1 to 4, e and f being from 1 to 6 and g bein~ fro~ 1 to 12), 3 - O.Z. 0050t40388 as well as the salts thereof with phy~iologically tol ra-ted acid~.

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

Particularly preferred physiologically tolerated acid~
are: hydrochloric acid, citric acid, tartaric acid, lactic acid, phosphoric acid, methanesulfonic acid, acetic acid, formic acid, maleic acid, fumaric acid, malic acid, succinic acid, malonic acid, ~ulfuric acid, L-glutamic acid, L-a~partic acid, pyruvic acid, mucic acid, benzoic acid, glucuronic acid, oxalic acid, a~cor-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 ~onnected together) and, in particular, have a disulfide bridge (G = H, amins-protective group;
Z = OH, NH2~ carboxyl-protective group; N + Q = -(CH2)C-S-S-(CH2~ d- ) or a 8 ide chain bridge (G = H, amino-protec-tive group, Z = OH, NHz, carboxyl-protective group, M + Q
= -(CH2).-NH-CO-(CH2)~- or -(CH2).-NH-CO-(CH2)s-NH-CO-(CH2)~-) or be linked head-to-tail (G ~ Z = covalent bond or -CO-(CH2).-N~-).

The no~el compound~ can be prepared by conventional methods of peptide chemistry.

Thus~ the peptides can be constructed sequentially from amino acids or by linking together suitable smaller peptide fragments. In the ~equential construction, the peptide chain i8 extended ~tepwi~e, by one amino acid e~ch time, ~tartinq at the C terminus. In the ca~e of coupling of fragment~ it i8 possible to link together - 4 - O.Z. 0050/40388 fragments of di~ferent lengths, these in turn being obtainable by sequential construction from amino acid~ or coupling of other fragment~. The cyclic peptides are obtained, after synt~.esis of the open-chain peptides, by a cyclization reaction carried out in high dilution.

In the case both of sequential con~truction and of fragment coupling it i8 necessary for the building blooks to be linked by formation of an amide linkage. Enzymatic and chemical method~ are suitable for this.

lD Chemical method~ for forming amide linkages are dealt with in detail by N~ller, Methoden der Organi~chen Chemie (Method~ of Organic Chemi~try) 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, Rockford, 1984; Bodan~zky, Rlausner, Ondetti, Peptide Synthesi3, pp 85-128, John Wiley ~ Son~, New York, 1976 and other ~tandard W0~8 of peptide chemistry.
Particularly preferred are the azide method, the symmetr-ical and mixed anhydride method, active estars generated in 8itu or preformed and the formation of amide linkage~
u~inq coupllng reagent~ (activator~ particular dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), l-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (E~DQ), 1-athyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (~DCI), n-propanepho~phonic anhydride (PPA), ~,N-bis(2-oxo-3-oxazolidinyl)~midopho~pho~yl chloride (BOP-Cl), diphenylpho~phoryl azide (DPPA), Castro' 8 rengent (BOP), O-benzotri~zolyl-N, N, ~ ', N '-tetra-methyluronium salt~ ( B TU), 2,5-diphenyl-2,3-dihydro-3-oxo-4-hydroxythiophene dioxide (Steglich'~ reagent;
HOTDO) and l,l'-carbonyldi~midazole (C~I~. The coupling re~gents csn be employed alone or in combination with additives ~uch as N,N'-dimethyl-4-aminopyridin~ (DMAP), N-hydroxybenzotriazole (BOBt), N-hydroxybenzotriazine (HOOBt)~ N-hydroxysuccinimide (HOSu) or 2-hydroxy-0 ~ 8 5 - O.Z. 0050/40~88 pyridine.

Wherea~ it i~ normally possible to di~pense with protec-tive groups in enzymatic peptide synthe~i~, for chemical synthe~i~ it i8 nece~sary for thera to be rever~ible S protection of the reactive functional group3 which are not involved in the formation of the amide linkage on the two reactant~. Three conventional protective group technique~ are ~referred for chemical peptide ~ynthe~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 i~ identified. The side-chain protective group~ on the trifunction~l amino acids are cho~en 80 that they are not nece~arily elimin-ated together with the -amino protective group. A
detailed review of amino acid protective groups i~ given by ~ller, ~ethoden der Organischen Chemie Vol XV/l, pp 20-906, Thieme Verlag, Stuttgart, 1974.

The building blocks used to con~truct the peptida chain can be reacted in ~olution, in su~pension or by a method similar to that described by Nerri~ield in J. Amer. Chem.
Soc. 85 (1963) 2149. Particularly preferred methods are tho~e in which peptid~ are constructed ~equentially or by fragment coupling by uae of the Z, Boc or F~oc protec-tive group technique, în which case the reaction take~place in 801ution, a~ well as those in which, similar to the Merrifield technique, one reactant i8 bound to an insoluble polymeric support (also called ra~in herein-~fter). Thi~ typically entail~ the peptide being con-~tructed sequentially on the polymes$c support, by u~e ofthe Boc or Fmoc protective group technique, with the growing peptide chain being covalently bonded aS the C terminu~ to the insoluble re~in particle~ (cf. Figure~
1 and 2). Thi~ procedurs allows rengent~ and byproducts to be removed by filtration, and thus recry~tallizat~on 2~3~)50~8 - 6 - O.Z. 0050/40388 of intermediate~ iq superfluous.

The protected amino acids can b~ bonded to any suitable polymers which merely need to be insoluble in the sol-vents used and to have a ~table physical form which allows easy filtration. The polymer must contain a functional group to which the fir~t protected amino acid can be firmly linked by a covalent bond. A wide variety of polymer~ is suitable for this purpose, for example cellulo~e, polyvinyl alcohol, polymethacrylate, sulfon-ated polystyrene, chloromethylated copolymer of ~tyreneand divinylbenzene (M~rrifield re in), 4-methylbenz-hydrylamine-re~in (MBHA-re~in), phenylacetamidomethyl-resin (Pam-resin), p-benzyloxybenzyl alcohol-re~in, benzhydrylamine-resin (BHA-re~in~, 4-hydroxymethyl-benzoyloxymethyl-resin, the resin used by Breipohl et al.
(Tetrahedron Lett. 28 (1987) 565; from ~A~HEM), HYCRAN
re~in (from ORPEGEN) or SASRIN re~in (from BACHEM~.

Solvents suitable for peptide synthesis in solution are all tho~e which are inert under the reaction condition~, in particular water, N,N-dimethylformamide (DMF), dimethyl ~ulfoxide (DMSO), acetonitrile, dichloromethane (DCM), 1,4-dioxane, tetrahydrofuran (TH~), N-methyl-2-pyrrolidone (NMP) and misture~ of the ~aid solvent~.
Peptide synthes$a on polymeric support~ can bs carried out in all inert organic solvents which di~solve the aDino acid derivatives u~ed; however, ~olv~nts which al~o have r~sin-sw611ing properties are prefarred, such as DMF, ~X, ~ , acetonitrile and DMSO, as well as mixtur~
of th~e ~olvent~.

After the peptide ha~ been ~ynthe~iz~d it i8 cleaved off th~ polymeric ~upport. Th~ cl~avage condition~ for the various types of re~ins are disclo~ed in the literature.
The clsa~age reactions mo~t commonly use acid and palladium cataly~i~, in particular cleavage in anhydrous 200~058 - 7 - O.Z. ~050/40388 liquid hydroger. fluoride, in anhydrous trifluoromethane-sulfonic acid, in dilute or concentrated trifluoroacetic acid or palladium catalyzed cleavage in THF or ~HF-DC~
mixture~ in the presence of a weak base such a~ morpho-line. The protective groups may, depending on the choicethereof, ~e retained or likewise cleaved off under the cleavage conditions. Partial deprotection of the peptide may also be worthwhile if the intsntion is to carry out certain derivatization reactions or a cyclization.

Some of the novel peptides have good cytoto~ic proper-tie~. Some other~ of the peptides have high affinity for the cellular TNF receptor without, however, having cytotoxic activity. They are therefore TNF antagoni~ts.
They compete with natural TNF for binding to the cellular TNF receptor and thu~ suppress the TNF effect. The novel peptides are valuable drugs which can be employed for treating neoplastic diseases and autoimmun~ di~ea~e~ a~
well as for controlling and preventing infections, inflammation~ and transplant re~ection reaction~. Simple experiments can be used to elucidate the mode of action of the indiv$dual peptides. The cytotoxicity of the peptide is determined by incubating a TN~-sen~itive cel~
line in the presenc2 of the peptide. In a qecond experi-mental approach, the cell line i~ incubated wi~h the relevant peptide in the presence of a lethal amount of TNF. It ~ 8 po~sible in thi~ way to detact the TNF-antagonistic effect. In addition, the affinity of the peptide for the cellular TWF receptor is determined in an in vitro binding exp~riment.

The following test sy~tems wsre used to characterize the a~oni~tic and antagonistic effects of the novel peptidess I. Cytotoxicity tQst on TNF-sensitive indicator cells, II. Cytotoxicity antagonism test on TNF-sensitive indicator cells, - 8 ~ O.Z. 0050/40388 III. Competitive receptor-binding te~t on indicator cell~
expre~sing TNF receptor.

I. Cytotoxicity test The agonistic e~fects of tha novel peptide~ are asse~sed on the basis of their cytotoxic effect on TNF-sensitive cells (e.g. L929, MCF-7, A204, U937).
The test with L929 and MCF-7 was carried out as follows:

1. 100 ~1 of culture medium containing 3 to 5 x 103 freshly tryp inized, exponentially ~rowing, L929 cell~ (mouse) or MCF-7 cell~ (human) were pipetted into the well8 of a 96-well flat-bottom culture plate. The plate wa~ incubated at 37C
o~ernight. The air in the incubator was saturated with water vapor and contained 5% CO2 by volume.

The L929 culture medium contained 500 ml of lx Earl~'~ NæN (Boehringer Mannheim), 50 ml of heat-inactivated (56-C, 30 min) fetal calf serum (FCS), 50 ml of L-glutAmine (200 mN~, 5 ml of lOOx non-e~sential amino acids, 3 ml of lM HEPES
buffer pH 7.2,and 50 ml of gentamicin (50 mg/ml).

The NCF-7 culture medium contained 500 ml of lx Dulbecco's M~ (Boehringer Mannh~im), 100 ml of heat-inactivated (56C, 30 min) FCS, 5 ml of ~-glutamine and 5 ml of lOOx non-es~Qntial amino aoids.

2. Th~ next day 100 ~1 of the peptide solution to be t~3t~d wero adted to the cell culture~ and ~ub~ected to serial 2-fold dilution. In addition, 80~e cell control~ (i.e. cell culture~ not tre~ted with peptide dilution~ and 80m~ rhu-TWF
control~ (i.e. cell culturs~ treated with zaososs - 9 - O.Z. 0050/40388 recombinant human TNF) were al~o made up. The culture plate was incubated at 37C in an atmo-sphere of air saturated with water vapor and containing 53 CO2 by volume for 48 h.

3. The percentage of survivin~ cell~ in the cultures treated with peptide dilution was determined by ~taining with crystal violet. For this purpose, the lLquid was removed from the well~ of the test plate by tapping it. 50 ~1 of cry~tal violet ~olution were pipetted into each well.

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

~.75 g of crystal violet 1.75 ~ of NaCl 1~ 161.5 ml of ethanol 43.2 ml of 37S formaldehyde water ad 500 ml The crystal violet ~olution was left in the wells for 20 min and then likewi e re~oved by tapping.
Ths plate~ were then wa~hed 5 times by imm~rsion in water in order to remove dye not bound to the cells. The dye bound to the cells was extracted by adding 100 ~1 of re~gent solution (50% etha-nol, 0.1% gl~cial acetic acid, 49.9~ watar) to e~ch well.

4. The plates were shaken for 5 min to obtain a ~olution of uniform color in each well. The surviving cells were determined by mea~uring the extinction at 540 nm of the colored solution in the individual wells.

5. Sub~equently~ by relat~ng to the cell control, ;~OO;~iOS8 - 10 - O.Z. 0050/40388 the S0% cytotoxicity value was defined, and the reciprocal of the 4ample dilutLon which re~ulted in 50% cytotoxicity was calculated as the cyto-toxic activity of the te~t ~mple.

II. Cytotoxicity antagoni~m test The antagonistic effect of the peptide~ was asse6sed on the basi~ of their property of antagonizing the cytotoxic effect of rhu-TNF on TNF-sensitive cells (e.g. L929, MCF-7, A204, U937~. Tha cytotoxicity antagonism test with L929 and MCF-7 cells was carried out a~ follow~:
1. 100 ~1 of culture mediu~ containing 3 to 5 x 103 fre~hly trypsinized, exponentially growing, L929 cells (mouse) or MCF-7 cell~ (human) were pipetted into the wells of a 96-well flat-bottom culture plate. The plate was incubated at 37C
overnight. The air in the incub~tor was ~aturated with water vapor and contain~d 5% COz by volume.

The L929 culture medi~m contained 500 ml of lx Earl~'s MEN (Boehringer Mannheim), 50 ml of heat-inactivated (56~C, 30 min) FCS, 5 ~1 of L-gluta-mine (200 mM), 5 ~1 of lOOx non-e~ential amino acid~, 3 ml of lM HEPES buffer pH 7.2, and 500 ~1 of qentamicin (50 mg/ml).

The NCF-7 culture mediu~ contained 500 ml of lx Dulb~cco's M~M (Boehringer Mannheim), 100 ml of heat-inactivated (56~C, 30 ~ln) FCS, 5 ml of L-glutamine (200 m~) and 5 ml of lOOx non-e~s~nti~l a~ino acid~.

2. The n~xt day 100 ~1 of the peptide ~olution to be t~ted w~r~ added to the cell cultures and sub~ected to ~arial 2-fold dilution. Then, 100 ~1 of a rhu-TNP dilution $n culture medium, which ~C~05058 ~ O.Z. 0050/40388 dilution had an 80-100~ cytotoxic effect in the final concentration in the cell culture, w~re added to these cell cultures. In addition, some cell control~ (i.e. cell cultures not treated with peptide solution or with rhu-TNF solution) and some rhu-TNF control~ (= cell cultures treated only with rhu-~NF ~olutionJ were also made up. The culture plate was then incubated at 37C in an atmosphere of air ~aturated with wa~er vapor and containing 5% C02 by volume for 48 h.

3. The percentage of ~urviving cells in the culture~
treated with ~ubstance dilution wa~ determined by staining with crystal violet. For this purpo3e, the liguid was removed from the well~ of the te~t plate by tapping it. 50 ~1 of crystal violet solution w~re pipetted into each well.

The crystal violet ~olution had the composition ~pecified in I.3 The cry~tal violet solution was left ~n the wells for 20 min and then likewi3e removed by tapping.
ThR plates were then washed 5 times by immer~ion in water in order to remove dye not bound to the cells. Th~ dye bound to the cells wa~ extracted by adding 100 ~1 of reagent solution (50% etha-nol, 0.1% gla~ial ace~ic acid, 49.9S water) to each well.

4. ~he plates were shaken for 5 min to obtnin a ~olut~on of uniform color in each well. The ~urviving cell~ were determined by me~uring the sxtinction at 540 n~ of the colored 801ution in the individual wells.

5. Subsequently, by relating to the cell control and - 12 - O.Z. 0050/40388 the rhu-TNF control, the 50~ antagonism value was defined, and the sample concentration which resulted in 50% antagonism of rhu-TNF cytotox-icity at the rhu-TNF concentration u~ed wa~
S calculated as antagoni3tic activity of the sample tested.

III. Co~petitive receptor-binding test Both che agoni3tic and antagonistic effect~ of peptide~ ara conditional on the latter binding to the TNF receptor. This means that peptide~ with an agonistic or antagoni~tic effect compete with rhu-TNF for binding to the TNF r~ceptor on TNF-sensitivQ indicator cells (e~g~ ~937). The comp~ti-tive receptor-binding test wa~ carried out a~
follows~

1. 100 pl of medium containing variou~ concentra-tion~ of the peptide to be te~ted and of rhu-~NF
(- control) were pipetted into the reaction vessel~. The medium comprised 500 ml of PBS
(Boehringer Mannheim), 10 ml of heat-inactivated (56C, 30 min) FCS and 100 mg of sodium azide.

2. Sub~equently, lO0 pl of medium containing 1 ng of ~ labeled rhu-TNF (Bolton lactoparoxidase method) were placed in the reactlon ve~els and mixed. The non-sp~ciflc binding (NSB) was det~r-mined ~y mixing in the reaction vesselR the ~I-labeled rhu-TNF (1 ng of ~I-rhu-TNF in 100 pl of mQdiu~ with a 200-fold exce~ of unlabeled r~u-TNF (200 ng of rhu-TNF in 100 ~l of medium).

3. Then 100 pl of medium centaining 2 x 10 U937 cells (human) were pipett~d into tha reaction ve~sel~ and mised. ~e reaction ve~sels (tast volu~e 300 pl) wars incubated at 0C for 90 min.

- 13 - O.Z. 0050/40388 The reaction mixtures were remixed after 45 min.

4. After the incubation the cells were centrifuged at 1800 rpm and 4C for S min, washed 3 tLme~
with medium and tran~ferred quantitatively into countin~ vial~, and the cell-bound radioactivity wa~ determined in a Clini gamma counter 1272 (LRB Wallac).

5. After the m0asurements had been corrected for the non-~pecific bindin~, ~he 50% competition value was defined by relation to the overall binding, and the ~ample concentration which led to 50%
competition of l251-rhu-TWF binding at the12~I-rhu-TNF concentration used was calculated a~ the competiti~e activity of the sample te~ted.

The Example~ which follow are intended to explain ths invention in more detail. The proteinogenous amino acid~
are abbreviated ln the Examples usi~g the conventional three-letter cod~. Other meaning~ are: A2d = ~-amino-adipic acid, Ab8 - 4-aminobutyric acid, Ac = acetic acid, Ade = 10-aminodecanoic acid, Ahp = 7-aminoheptanoic acid, Hcy = homocysteine, Hly = homoly~ine, Orn = ornithine, Dap = 2,3-diaminopropionic acid.

A. General procedure I. The pQptides claimed in cla~ 1 were synthe~ized u~ing ~tzndard methods of solid-pha~e peptide ~ynthe~is in a completely automatic model 430A
psptide ~ynthe~izer from APPLI~D ~IOSYSTEMS. Ths apparatus uses different synthe~is cycles for the ~oc and Fmoc protective group techniques.

aJ Synthe~is cycle for the Boc protective group technique 2QC)5058 - 14 - O . Z . 0050/40388 1. 30% trifluom~etic acid in DCM 1 ~c 3 ~
2. 5096 trifluoro~etic ~id in DCM 1 x 17 min 3. ~M washillg S x 1 min 4. 596 dii~let~lamine in DQ~ 1 x 1 ~
S 5. 5% diis~let~lamine in 2~P 1 ~ 1 min 6. NMe washing 5 x 1 min æid (activa~on by 1 equivale~t of DOC
and 1 ~valerrt of ~t in NMP/DC~);
p~ptide c~lir.~g (lst part) 1 x 30 min 8. Additic)n of nM50 to the rea~ti~ mixh~re until it coq~tain~ 2096 1~;0 }~y volu~e 9. P~de a~ (2"~ part) 1 x 16 min 10. ~itia~ of 3.8 eq!livalenl:s of dii~

11. P~ c~lis~ (3rd part) 1 x 7 min 12. rY'M wa~hing 3 x 1 mi~

13. If rea~tion i~ inc~lete, reeetit~
of c~1in3 (re~nl to 5.) 14. 109~ a~:etic ar~id~, 5% dii~l-e~l~minP in DCM 1 x 2 min 15. 10% a~etic ar~ydr~de in D~l x 4 min 16. DC~ wa~nq 4 x 1 ~in 17. ~n to 1.

b) ~is ~ycle f~ tl~ E~c protective grc~up te~mi~e 1. N~ ~ng 1 x 1 min 2. 20% pi~r~ n ~e 1 x 4 min 3. 20% pi~ridino in llMP 1 x 16 min 4. N~P wo~hiJlg S x 1 min 5. A~diti~n of preactivated pmt~ aoin~

wtide coupli~ 1 x 61 min 6. ~ w~hir~ 3 x 1 min 7. If reaction i8 i~1ete, reQetitiQn of ~ (~ to 5. ~

~30~0~8 - 15 -O.Z. 0~50/4038~
8. 10% ~ ridb Ln NMP1 x 8 min 9. NNP washing 3 x 1 min lO. ~n to 2.

II. Working up of peptide-resin~ obtained as in Ia The peptide-resin obtained a~ in Ia was dried under reduced pressure and transferred into a reaction vessel of a Teflon HF apparatus (from PENINSULA).
Addition of a scavenger, preferably ani~ole (1 ml/g of re~in), and of a thiol, in the case of tryptophan-containing peptides, to remove the indole formyl group, preferably ethanedithiol (0.5 ml~g o~ resin)~
wa~ follewed by conden~ation in of hydrogen fluoride (10 ml/g of re~in) while cooling with liquid N2. The mixture was allowed to warm to 0C, and was ~t~rred lS at thi3 temperature for 45 min. The hydrogen fluor-ide was then ~tripped off under reduced pre~surQ and the residue wa~ washed with ethyl acetate in order to remo~e remaining scavenge~. The peptide wa~
extracted with 30S ~trength acetic acid and filtered, and the filtrate was freeze-driQd.

To prepare peptida hydrazide~, the peptide-resin (pam- or Merrifield re~in) wa~ su~pended in DMF
~15 ml/g of re~in), hydrazine hydrste (20 equiva-lont~) wa~ added, and tha mixture wa~ stirred at room temperature for 2 days. To work up, the re~in was filtered off and the filtrate wa~ evapor2ted to dryness. The residue wss cry~tRlli~ed from DMF/Et2O
or NeOH~t2.0-III. Working up of the peptide-resins obt~ined as in Ib The peptide-resin obtained a~ in Ib was dried under reduced pre~sure and ~ub~equ~ntly ~ub~ected to one of the following cleavage procedure~, depending on the ~mino acid composition (W~de, Tregear, Hownrd Florey Fmoc-Work~hop ~anual, Melbourne 1985)~

)50~8 16 - O.Z. 0050/40388 Peptide containing Clea~age condition~

Arg(Mtr) Met Trp TFA Scavenger Reaction Time S
no no no 95% 5~ H2O 1.5 h yes no no 95% 5~ thioanisole 2 3 h no ye8 no 95% 5~ ethyl methyl 1.5 h sulfide no . no yes 95~ 5~ ethanedithiol/1.5 h anisole (1:3) no yes yeR 95% 5~ ethanedithiol/1.5 h anisole~ethyl methyl sulfide (ls3:1) yes yes yes 93S 7~ ethanedithiol/~ 3 h anisole/ethyl methyl sulfide (1:3:3) The su~pen~ion of the peptide-re~in in the suitable TFA mixture wa~ stirred st room temperatur~ for the stat~d time and then the resin was filtered o$f and washed with TFA and with DC~. The filtrate and the wa~hing~ were extensiv~ly concsntrated, and the pep~ide was precipitated by addition of diethyl eth~r. The mixture wa~ cooled in an ica bath, and the pracipitate was filtered of~, taken up in 30%
acetic ~cid and freeze-dried.

IV. Purifi~ation and charactorization of the peptides Purification was by gel chromatography (SEPHAD~X
G-10, G-15/10~ HOAc; SEPHAD~X~ LH20/MeOH) and aub-s~quent medium pre~sure chromatography (~tationary phases HD-SIL C-18, 20-45 ~, 100 A; mobile pha~e:
gradient with a = o.l~ TFA/NbOH, A ~ 0.1~ TFA/H20).

2~3C)5~58 - 17 - O.Z. 0050~4038~
The purity of the final product~ was determined by analytical HPLC (~tationary phase: 100 x 2.1 mm VYDAC C-18, 5 ~, 300 ~; mobile pha~e = CH3CN/H20 gradient buffered with 0.1~ TFA, 40C). Charac-terization wa~ by means of amino acid analy~i~ and fa~t atom ~omhardment mass spectrometry.

B. Specific procedure~

Ac-Leu-Glu-Lys-Gly-A~p-Arg-NH2 1.47 g of Boc-Arg(Tos)-MBHA-re~in (~ub3titution O.34 mmol/g~, corre~ponding to a batch size of 0.5 mmol, were reacted as in AIa with 2 mmol each of Boc-Asp(OChx)-OH Boc-Glu(OChx)-OH
Boc-Gly-OH Boc-Leu-OH
Boc-Ly8(Cl-Z)-OH

After the synthasi~ was complete, the N terminus was acotylated (steps 1-6 and 14-16 as in AIa). The peptide-re~in wa~ dried under reduced pre~ure; the yield was 1.87 g.

The re~in obtained ~n this way wa~ sub~e~ed to HF
cleavage as in AII. The crude product (181 mg) was purified by gel flltration (SEPHADEX G-10) and medium pro~sure chromatography (cf. AI~; 5-20~ A; 0.25% min~l).
78 mq of pure product were obtained.

H-Phe-Gln-Glu-Lys-Gly-Asp-~rg-Leu-OH

0.47 g of F~oc-Leu-p-alkoxyben~yl alcohol-resin (sub~ti-tution 0.53 mmol~g)~ corre~ponding to a batch ~ize of 0.25 mmol, ware reacted a~ in AIb with 1 lol each of ;~0~1058 - 18 - O.Z. 0050/40388 Fmoc-Arg(~tr)-OH Fmoc-Glu(OtBu)-OH
Fmoc-Asp(OtBu)-OH Fmoc-Gln-OH
Fmoc-Gly-OH Fmoc-Phe-O~
Fmoc-Lys(Boc)-OH

S After the synthesis was complete, the peptide-resin underwent N-terminal deprotec~ion (step~ 2-4 as in AIb) and drying under reduced pre3sure; the yield was 0.75 g.

The crude peptide (212 m~) obtained after TFA cleavage a~
in AIII was purified by gel filtration (SEPHADEX G-10) and medium pre~ure chromatography (cf. AIV; 5-25% A;
0.25~ min~l). 136 mg of pure product were obtained.

Th~ following can be prepared in a ~imilar manner to Examples 1 and 2:

3 H-Glu-Lys-Gly-Asp-Arg-OH

4 Ac-Glu-Lys-Gly-Asp-Arg-OH

Ac-Glu-Lys-Gly-Asp-Arg-NH2 6 H-~ou-Glu-L~s-Gly-~sp-Arg-OH

7 H-Leu-Glu-Ly5-Gly-A5p-Ar9-NH 2 8 H-Gln-Glu-Lys-Gly-Asp-Arg-OH

9 Ac-Gln-Glu-Lys-Gly-~sp-Arg-OH

10. H-Gtn-Glu-Lys-Gly-~sp-~r9-NH2 Il Ac-Gln-Glu-Lys-Gty-Asp-~r9-NH2 IZ H-Ph~-Gln-Glu-Lys-Gly-Asp-Arg-OH
13 Ac-P~-Gln-Glu-Lys-Gly-Asp-~rg-OH
14 H Phc-Gln-Glu-Lys-Gly-Asp-Arg-NH2 ~c-Ph~-Gln-Glu-L~s-Gl~-Asp-~rg-NH2 16 Ac-Ph--61n-Glu-L~s-Gly-Asp-Arg-L~u-NH2 17 H-Lcu-Phe-G1n-Glu-Lys-Gly-~sp-Arg-Lou-OH
18 Ac-L~u-Ph--Gln-Glu-Lys-Gly-~sp-Arg-Lcu-NH2 19 H-Gln Leu-Phc-Gln-Glu-Lys-Gl~-Asp-~rg-L~u-S~r-OH
Ac-Gln-L~u-Ph~-Gln-Glu-~ys-Gly-~sp-Arg-~u-S~r-NH2 21 H-L~u-61u-Lys-Glg-Asp-L~s-OH
22 ~c-Lcu-Asp-Lys-Sly-~sp-Arg-NH2 23 Ac-Lcu-61u-0-Lys-Gly Asp-Arg-NH2 24 Ac-P~-G1n-Glu-Gln-Gly-~sp-~rg-NH2 2S H-Gln-L~u-T~r-~ln-61u-L~s-Gly-~sp-~rg-L~u-S-r-OH

2~0~0~8 - 19 - O.Z. 0050/~0388 Ac-Hcy-Gln-Leu-Glu-Lys-Gly-Asp-Arg-Hcy-NHz 0.41 g of Boc-Hcy(pMB)-NBHA-resin (substitution 1.24 mmol/~), corresponding to a batch size of 0.5 mmol, waB reacted a~ in AIa with 2 mmol each of Boc-Arg(Tos)-OH Boc-Glu(OChx)-OH
Boc-Asp(OChx)-OH Boc-Leu-OH
~oc-Gly-OH Boc-Gln-OH
Boc-Lys(Cl-Z)-OH Boc-HCy-(pMB)-OH.

After the ~ynthe~is was complete, the N terminu~ was acetylated (~teps 1-6 and 14-16 a~ in AIa). The peptide-resin was dried under reduced pressure; the yield was 1.1 g.

The re~in obtained in thi~ way was sub~ected to HF
cleavaga as in AII. The freeze-dried crude product wa~
taken up in 2 1 of 0.1~ strength acatic acid, and the pH
wa~ then ad~uated to 8.4 with aqueous ammonia. Under an argon atmo~phere, 0.01 n R3~Fe(CN)~] solution was slowly added dropwi~e until the yellowi~h-green color persisted for at least 15 min. The mixture wa~ then stirred for 1 h and acidified to pH 4.5 wi~h glacial acetic acid, and 15 ml of an aqueou~ ~uspension of an anionic exchanger (BIORAD 3 x 4A, chloride form) were added. After 30 min, the ion exchan~er resin was filtered off and the filtrate was concentrated to 100 ml in a rotnry evaporator and ~ubsequently freeze-dried.

All the ~ol~ents had previously been saturated with nitrogen in order to pre~ent any ox~dation of the free cy~teins resldue~.

The crude product wa~ purified by gel chro~atography XC~0~058 - 2~ - O.Z. 0050~40388 (SEPHADEX G-15) and medium pressure chromatography tcf.
AIV; 30-60% A; 0.25~ min~1). 15 mg of pure product wQre obtained.

The following can be prepared in a similar manner to Example 26 ~Pam-re~in was u~ed to prepare the peptide acids)s 27. H-C~s-Lys-Gl~-Asp-Cys-O~
28. H-Cys-~lu-Lys-Gly-~sp-Arg-Cys-OH
29. Ac-Cys-61u-Lys-Gly-Asp-~rg-Cys-~Ha 30. H-Hcy-Glu-Lys-Gly-Asp-Arg-Hcy-OH
31. Ac-Hcy-&lu-Lys-61y-Asp-Arg-Hcy-NH2 32. Ac-Cys-~eu-Glu-Lys-GI~-Asp-~rg-Cys-NH2 33. Ac-Ucy-Leu-Glu-Lys-Gly-Asp-~rg-Hcy-NH2 34. Ac-Cys-Glu-~ys-¢ly-~sp-Arq-Leu-Cys-NH2 35. Ac-Hcy-Glu-Lys-Gly-Asp-Arg-Leu-Hcy-NH2 36. Ac-Cys-Leu-Glu-Lys-Gly-Asp-Arg-Leu-Cys-NH2 37. Ac-Hcy-Leu-Glu-LyS-Gly-Asp-Arg-Leu-Hcy-NH2 38. H-Cys-Gln-Leu-Glu-Lys-Gly-~sp-~rg-Cys-OH
3~. Ac-Cys-Gln-Leu-Glu-Lys-Gly-Asp-Arg-CyS-NH2 40. ~-Hcy-Gln-Leu-Gtu-Lys-Gly-Asp-Arg-Cys-OH
41. Ac-Hcy-Gln-Leu-Glu-Lys-61y-Asp-~rg-Cys-NH2 42. H-Cys-Gln-Leu-Glu-Lys-Gly-Asp-~rg-Hcy-OH
43. H-Hcy-Gln-Leu-Glu-Lys-Gly-Asp-Arg-Hcy-OH
44. Ac-Hcy-Gln-Leu-Glu-Lys-Gly-Asp-~rg-Hcy-NH2 45. Ac-Gln-Leu-Cys-Lys-Gly-Asp-Cys-Leu-S~r-NH2 46. Ac-Hc~-Glu-Arg-Gl~-ASp-Hcy-NH2 47. Ac-Phe Gln-Hc~-Glu-Lys-Gly-~sp-~cy-Leu-Sor-~l--N~2 '~ O S 0 5 8 - 21 - O.Z. 0050/40388 48. H-Cys-Leu-GIu-Lys-GIy-Asp-Lys-cys-oH
49. H-Cys-Leu-Glu-O-Lys-Gly-Asp-Arg-Cys-OH
50. Ac-Cys-Leu-Asp-Lys-G~y-Asp-Arg-Leu-Cys-NH2 51. Ac-Cys-Bal-Gtn-Leu-Glu-Lys-Gly-Asp-Arg-Cys-N~2 52. Ac-C~s-Gly-Glu-Lys-Gly-Asp-Arg-Cys-NH2 53. Ac-Cys-G1u-Lys-Gly-Asp-Cjs-NH2 54. Ac-Cys-Gl~-Glu-Lys-G1~-Asp-~rg-Gly-Cys-NH2 Ac-A~p-Glu-Lys-Gly-ARp-Arg-Lys -NH2 1 g of resin de~cribed by Breipohl et al. (from BACHE~), corresponding to a batch ~ize of 0.5 mmol, was r~acted a~
in A~b with 2 mmol each of Fmoc-Ly~(Boc)-OH Fmoc-Ly~(2)-O~
Fmoc-Arg(To~)-OH Fmoc-Glu(OBzl)-OH
Fm~c-A~p(OChx)-OH Fmoc-A~p(OtBu)-OH
Fmoc-Gly-OH

After the synthe~is was complete, the N terminus wa~
acetylated (~teps 2-4 and 8-9 a~ in AIb). The peptide-re~in w~ dried under reduced prsssure; yield 1.64 g.

~he crude product (592 mg) obtained after TFA cle~vage as in AIII wa8 dis~olved in 500 ml of dega~ed DMF; to thi~
solution w~re added at -15-C 61.1 mg ~0.5 ....ol) of DMAP
and 1.92 g (40 m~ol) of ~DCI. The mixture wns then ~tored at -15-C for 2 days, at 0'C for 2 days and at RT for 2 day~. To worX up, th~ mixture was evaporated under reduced pressure, and the residue wae digested with water. The precipitated pept~-de wa~ filtered o~f, washed zoososa - 22 - O.Z. 0050/4~388 ~everal tLme~ with ~ citric acid and water and, after drying (P20s), purified by gel chromatography (SEPHADEX-LH 20). The isolated and well dried monomer (183 mg) was sub~ected to HF cleavage as in AII and ~ubsequently S purified by medium pressure chromatography (cf. AIV; 20-40~ A; 0.25% mLn~l). 121 mg of pure product were obtained.

EXA~PLE 56 Ac-Asp-Glu-Lys-Gly-A~p-~rg-~ ~-OH

1.47 g of Fmoc-Ly~(Boc)-Nerrifield-resin (substitution 0.34 mmol/g), corre3ponding to a batch size of 0.5 mmol, were reacted as in AIb with 2 mmol each of Fmoc-Arg~Tos)-OH F~oc-Ly~(Z)-OH
Fmoc-A~p(OChx~-0~ Fmoc-Glu(OBzl)-OH
Fmoc-Gly-O~ Fmoc-A~p(Ot~u)-OH.

~he N terminu~ wa~ acetylated (steps 2 4 and 8-9 as in AIb).

The t-butyl and Bo~ protective qroups were sub~equently cleaved off (step~ 1-6 and 8-9 a~ in AIa). The cycliza-tion on the resin took place in NNP with the addition o~
O.89 g of BOP and 0.87 ml of dii~opropylethylamine (48 h). The y$eld was 1.85 g.

The crude product obtained after HF cleavage a~ in AII
was pur~fied by ~el filtration (Sephadex G-15) and medium pressure chromatography (cf. AIV; 15-35% A; 0.25~ min~l).
14 mg of pure product were obtained.

The following can be prepared in a ~milar manner to ~xample~ 55 and 56s ~1~05058 - 23 - O.Z. OOSO/40388 57. Ac-Glu-L~s-Gly-Asp-Ly5-N~2 58. H-Glu-Lys-Gly-ASp-Lys-OH
59. Ac-Glu-Lys-Gly-Asp-Arg-Lys-NH2 60. Ac-G~u-Lys-Gly-Asp-Arg-H~y-NH2 61. Ac-Glu-Gtu-Lys-Gty-Asp-~rg-~ys-NH2 62. Ac-Glu-Glu-Ly5-Gly-A5p-Arg-H~y-NH2 63. Ac-Asp-Glu-Lys-Gty-Asp-Arg-Ljs-NH2 6~. Ac-Lys-Glu-Lys-Gly-Asp-Arg-Asp-NH2 65. Ac-Hly-Glu-Lys-Gly-Asp-Arg-Asp-NH2 66. Ac-Orn-G1u-Lys-61y-Asp-Arg-Asp-NH2 67. Ac-Lys-Glu-Ly5-Gly-Asp-Arg-Glu-NH2 68. Ac-Lys-Leu-Glu-Lys-Gly-Asp-Ar9-Gtu-OH
69. Ac-Lys-Leu-Glu-Ly5-Gly-ASp-Arg-Glu-NH2 70. Ac-Gtu-L~u-Glu-Lys-Gly-Asp-Arg-Lys-NH2 71. Ac-Lys-~u-Glu-Lys-Gly-~sp-~rg-Leu-Giu-NH2 72. Ac-Lys-61u-Ly5-6t~-A5p-Arg-Leu-61u-NH2 73. Ac-6tu-61u-Lys-61y-Asp-~rg-Lcu-Lys-NH2 74. ~c-Ph~-61n-G ~ In-Ly5-Gly-~5p-Arg-Lys-S-r-~la-NH2 ~ 1 75. Ac-Asp-Gtu-Arg-61y-Asp-Arg-Hty-NH2 76. Ac-~ad-Glu-Ly5-61y-~sp-Arg-Orn-NH2 77. Ac-Lys-Glu-Lys-Gty-Asp-Lys-Giu-HH2 78. H-L~S-Glu-Lys-Gly-Asp-Lys-Glu-OH

2 ~ ~ ~ 0~ ~

- 24 - O.Z. 0050/40388 rhsu-Glu-Ly -Gly-Asp-Aocl 0.92 g of Fmoc-Gly-p-alkoxybenzyl alcohol-resin t~ubstit-ution 0.53 mmol/g), corresponding to a batch size of 0.5 mmol, was r~acted as in AIb with 2 mmol each of Fmoc-Lys(Z)-OH Fmoc-Aoc-OH
Fmoc-Glu(OChx)-OH Fmoc-Asp(OCHx)-OH
Fmoc-Leu-OH

After the synthesi~ was complete, the peptide-resin wa3 dried under reduced pre~sure. The yield ~a~ 1.43 g.

The crude peptide obtained after TFA cleavage as in AIII
wa~ di~solved in 500 ml of dega~ed DMP and reacted and worked up aa in Exemple 55. 97 mg o~ pure product were obtained.

The following can be prepared i~ a ~milar manner to Example 79:

80. rD-Leu-Glu-Lys-Gl~-Asp-~r~
81. ~lu-Lys-Gly-~sp-Arg-Ade 82. rLeu-Glu-Lys-Gly-Asp-~rg-Leu 83. rL~u-Glu-Lys-Gly-Asp-Arg-Ahp 84. rL~u-Gtu-Lys-61y-Asp-Arg-Leu-Abs 8S.
86. rL~U-GtU-O--Y5-~lY-~SP-~r9 87. ~sp-~s-Gl~-Asp-Arg-~d~
88. ~L~-61u-~s-Gly-Asp-Lys-Ah~
89. r~-Glu-Lys-Gly-~sp-Arg-Le~

~305058 - 25 - O. Z . 0050/40388 90. rPhe-61n-Leu-S;lu-LyS-Gly-Asp-Arg-Leu-Ser-Gly 91. rPhe-61n-Leu-Gtu-Lys-Gly-Asp-Arg-Leu-Ser-Pal

Claims (8)

1. A peptide of the formula I
X-A-Gly-Asp-Y I
where A is Lys, Gln or Arg, 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 together 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, -C8H5, -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-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)e-CO-NH-(CH2)f or-(CH2)e-NH-CO-(CH2)g-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 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 aa claimed in claim 1, where G is hydrogen or an amino-protective group and Z is hydroxyl or amino or a carboxyl-protective group, and M and Q
together are a -(CH2)c-S-S-(CH2)d-bridge.

4. A peptide as claimed in claim 1, where G is hydrogen or an amino-protective group and Z is hydroxyl or amino or a carboxyl-protective group, and M and Q
together are -(CH2)?-NH-CO-(CH2)f- or -(CH2)?-NH-CO-(CH2)6-NH-CO-(CH2)f.

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

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

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

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