AU640739B2 - Peptides which inhibit blood coagulation, processes for the preparation thereof and the use thereof - Google Patents

Abstract

Peptides of the formula I A1-A2-A3-A4-A5-A6-A7-A8-A9-A10-A11-A12-A13-A14-A15 in which A1 is hydrogen, cysteine, acetylcysteine, one or two alkyl groups with 1-4 C atoms, an acyl group with 2-10 C atoms, an acyl group with 2-10 C atoms and another carboxyl group, or a protective group customary in peptide chemistry, A2 is a bond, Asn, Asp, Gln or Glu, A3 is a bond, Gly or Ala, A4 is Glu or Asp, A5 is Phe, Tyr, Trp, Pgl (phenylglycine) or Nal (naphthylalanine), A6 is Glu or Asp, A7 is Glu, Asp, Pro or Ala, A8 is Ile, Leu, Val, Nle or Phe, A9 is Pro or Hyp, A10 is Glu or Asp, A11 is Glu or Asp, A12 is Phe(SO3H) or Phe(PO3H2) (preferably in the p position) or Pgl(SO3H) or Pgl(PO3H2) (preferably in the p position), A13 is a bond, Leu, Ile, Val or Ala, A14 is a bond, Gln, Asn, Glu, Asp or Cys and A15 is Cys, Cys-amide, an OH group of the alpha-carboxyl group, free or esterified with a lower alcohol with up to 4 C atoms, which can also be in the form of the carboxamide functionality whose hydrogens can optionally be replaced by alkyl groups with up to 4 C atoms, and a process for the preparation thereof are described. These peptides inhibit blood coagulation and can be used as anticoagulants.

Description

COMMONWEALTH OF AUSTRA PATENTS ACT 1952-69 COMPLETE SPECIFICATION

(ORIGINAL)

Class Int. Class Application Number: Lodged: Complete Specification Lodged: *o 0 Related Art 0 Related Art

S..

Accepted: Published: of Applicant 0* Address of Applicant: Actual Inventor: dress for Service Aaddress for Service BEHRINGWERKE AKTIENGESELLSCHAFT D-3550 Marburg, Federal Republic of Germany WERNER STUBER WATERMARK PATENT TRADEMARK ATTORNEYS.

LOCKED BAG NO. 5, HAWTHORN, VICTORIA 3122, AUSTRALIA Complete Specification for the invention entitled: PEPTIDES WHICH INHIBIT BLOOD COAGULATION, PROCESSES FOR THE PREPARATION THEREOF AND THE USE THEREOF The following statement is a full description of this invention, including the best method of performing it known to US BEHRINGWERKE AKTIENGESELLSCHAFT 90/B 007 Ma 822 Dr. Ha/Bi Peptides which inhibit blood coagulation, proces -s for the preparation thereof and the use thereof The present invention relates to peptides which inhibit blood coagulation, processes for the preparation thereof and the use thereof as anticoagulants.

Anticoagulants are of great therapeutic relevance in the treatment of various disorders affecting blood coagu- 10 lation, such as disseminated intravascular coagulation, myocardial infarct and deep vein thrombosis. Currently employed for the therapy of these disorders are anticoagulants such as antithrombin III which is obtained from S"human plasma.

Recently a polypeptide from the leech (Hirudo medicinalis) composed of 65 amino acids has been tested as anticoagulant. However, the use of hirudin, as this "peptide is also called, is associated with various disadvantages. One disadvantage is the problem of the low availability of this substance. Possible difficulties may also derive from the relatively high molecular weight of this peptide, which means that there is a potential risk of antibody production.

S

It has been possible to circumvent these disadvantages by developing low molecular weight peptides as antic,agulants, which have a high degree of homology with the\ Cterminal region of hirudin. Peptides of this type are described in the application KP-A 0 276 014, EP 0 333 356 and in a publication by J.M. Maraganore et al., J. Biol.

Chem. 264, 8692-8698 (19R9).

It is evident therefrom that a peptide of the structure A3n-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-OH is of particular interest. A peptide whose tyrosine (Tyr) is 2 sulfated on the phenolic group detectably had particularly high activity. This moreover corresponds to native hirudin whose tyrosine at position 63 is sulfated.

Cyclic peptides are also of interest. These contain, in addition to the sequence just mentioned, the amino acid cysteine at the C- and N-terminus, which permits cyclization in the form of a disulfide. A disulfide bridge can also be replaced by other chemical functions, preferably a connection via an amide linkage.

10 The chemical nature of a sulfated tyrosine is that of an ester of sulfuric acid so that the linkage between the sulfur atom and the phenolic oxygen atom can be cleaved by hydrolysis. Peptides of this type have the disadvantage of a reduced anticoagulant activity.

Thus, according to the state of the art, attempts are being made to achieve the sulfation on tyrosine by a subsequent chemical reaction. For this purpose, the G unsulfated hirudin peptides are reacted with dicyclo- S hexylcarbodiimide and sulfuric acid in organic solvents.

120 The sulfation can also be achieved by reaction of the tyrosine-containing peptide with sulfur trioxide triethylammonium salt in pyridine or chlorosulfonic acid.

However, these reactions have the disadvantages that side •reactions Lan take place on phenylalanine or non-selective sulfation can take place when several tyrosine residues are present. This may also lead to large losses in yield.

Hence the object of the present invention was to eliminate these disadvantages and to prepare peptides with superior physical, chemical and physiological properties.

This object has been achieved, surprisingly, by replacing the amino acid tyrosine or Tyr(SO 3 H) in the peptides by the amino acid Phe(SOH) or Phe(PO 3

H

2 or Pgl(SO 3 H) (Pgl 3 denotes phenylglycine) or PgI(PO0H 2 In this connection, the sulfonate or phosphate group is preferably linked in the para position in the phenyl ring of the phenylalanine, but a linkage in the meta position is likewise possible.

Hence the invention relates to a peptide of the formula: A1-A2-A3-A4-A5-A6-A7-A8-A9-A10-All-A12-Al3-A14-Al5 in which Al is hydrogen, cysteine, one or two alkyl groups with 10 1-4 carbon atoms, an acyl group with 2-10 carbon atoms, an acyl group with 2-10 carbon atoms and another carboxyl group, or a protective group ,...,custorary in peptide chemistry, A2 is a bond, Asn, Asp, Gln or Glu, *'15 A3 is a bond, Gly or Ala, A4 is Glu or Asp, is Phe, Tyr, Trp, Pgl (phenylglycine) or Nal (naphthylalanine), A6 is Glu or Asp, *20 A7 is Glu, Asp, Pro or Ala, A8 is Ile, Leu, Val, Nle or Phe, A9 is Pro or Hyp, is Glu or Asp, All is Glu or Asp, '25 A12 is Phe(SO 3 H) or Phe(POH 2 (preferably in the p position) or Pgl(SO 3 H) or Pgl(P0 3

H

2 (preferably in the p position), A13 is a bond, Leu, Ile, Val or Ala, A14 is a bond, Gin, Asn, Glu, Asp or Cys and is Cys, Cys amide, an OH group of the alpha-carboxyl group, free or esterified with a lower alcohol with up to 4 carbon atoms, which can also be in the form of a carboxamide group whose hydrogens can optional- A Lly be replaced by alkyl groups with up to 4 carbon S, atoms.

4 If Al is the amino acid cysteine, the amino group can also be acetylated.

The peptides according to the invention are synthesized by methods which are sufficiently known Barany and R.B. Merrifield in "The Peptides" Gross and I. Meienhofer, The amino acids Phe(SO 3 H) or Pgl(SO 3 H) can be obtained by sulfonation of phenylalanine or phenylglycine, respectively, which are in the D or L or D,L, preferably in the L, form. A suitable sulfonating agent is sulfuric acid, which preferably also contains sulfur trioxide.

S These amino sulfonic acids were provided, by methods known from C.D. Chang et al., Int. J. Peptide Protein SRes. 15, 59 (1980), with a protective group on the amino "'15 group in order to be able to synthesize a peptide according to the invention. Suitable protective groups in this context are: Boc, Z, Bpoc, Ddz and, preferably, the Fmoc group.

The peptides were synthesized either by a peptide syn- 020 thesis method operating in solution and entailing known procedures Winsch in "Houben-Weyl, Synthese von Peptiden I" (Synthesis of Peptides), G. Thieme Verlag, Stuttgart (1974)) or by solid-phase methods which are likewise known (see above), in which case Fmoc chemistry '.25 was preferably used.

The sulfonate or phosphate group was employed unprotected in the synthesis.

In the solid-phase pept-Le synthesis, the peptide chain was synthesized on crosslinked polystyrene (1 divinylbenzene) (called resin hereinafter). The synthesis of peptides with free carboxyl groups made use of anchors based on alkoxybenzyl alcohol. Amide anchors (Int.

J. Peptide Protein Res. 34, 262-267, 1989) which produce 5 non-alkylated peptide amides were used for peptide amides. The incorporation of the individual protected amino acids was carried out in a repetitive pattern: introduction of the Fmoc-amino acid (or amide anchor)-resin into a completely automatic peptide synthesizer washing of the resin with DMF, dichloromethane or Nmethylpyrrolidone (about 15 ml/mg) elimination of the Fmoc group with 20 piperidine in dimethylformamide or N-methylpyrrolidone (preferably 1 x 3 min and 1 x 10 min) removal of the piperidine by washing with DMF, dichloromethane, N-methylpyrrolidone or an alcohol, preferably isopropanol *e coupling of the amino acid using a carbodiimide, preferably diisopropylcarbodiimide, where appropriate with the addition of HOBt, HOSu or with the use of BOP or TBTU, where appropriate with the addition of HOBt, preferably in DMF or in N-methylpyrrolidone.

6 •The sidechains of the trifunctional amino acids were o. protected as follows: Asp and Glu as t.-butyl ester S Hyp and Tyr as t.-butyl ether 0**25 Cys as trityl ether or tert.-butyl dicalfide.

In place of the Fmoc chemistry described above, these peptides can also be synthesized using the Boc strategy Stewart and J.D. Young "Solid Phase Peptide Synthesis" Pierce Chemical Co., 1984, pp. 71-95) because the sulfonate group is not damaged by repetitive use of trifluoroacetic acid, In the case of Fmoc chemistry, the peptides were eliminated from the resin using trifluoroacetic acid, preferably with the addition of a scavenger, and crystallized 6 using ether. After purification by reversed phase chromatography, their composition was confirmed by aminoacid analysis and FAB mass spectrometry.

When cysteine-containing peptides were prepared, in the case of Cys(Trt) the trityl protection was removed simultaneously with the peptide elimination as long as the elimination mixture contained an added thiol, preferably ethanedithiol.

When Cys(StBu) was employed, the S-tBu group was prefer- 10 ably removed after elimination of the peptide. Used for 0* this purpose were known methods, such as, for example, treatment with tri-n-butylphosphine or dithioth:eitol.

Dithiothreitol deprotection was preferably used.

The cyclization via S-S bridges could be carried out by known oxidative methods.

The cysteine-containing peptide was pireferably dissolved in a concentration of 0.1 to 0.001 mM in ammonium bicarbonate buffer (0.01 M) and shaken in the air for several O hours. The cyclization was followed by HPLC.

*20 Other cyclization methods, such as, for example, oxidations with iodine, for example in acetic acid, or K3[Fe(CN),] are likewise suitable for this purpose.

A possible alternative is the preparation by a method operating in solution, in which case individual fragments of the complete peptide are initially prepared. Condensation of individual protected amino acids to give peptide segments was carried out in solvents such as DMF and tetrahydrofuran or mixtures thereof. The coupling of the amino acids was effected using carbodiimides as in the case of solid-phase synthesis. Individual segments were then combined to give the complete peptides. After elimination of the protective groups, the peptides were likewise purified and characterized.

-7 The peptides were tested f or their activiLty in a functional assay.I The following specific peptides were p~epared,, but the contents of the invention are not confi'ecO to them: Abbreviations:

C

C

0C C I 0W a 0 be be 0CC

I

*IeOOS 0 0000 20 00 C 00 ee

C

see...

*25 00

C.

00 Asn L-asparagjine Asp L-aspartic acid Cys L-cyS4,eine Gin L-giutamine Glu L-glutamic acid Gly glycine Ala L-alanine Tyr L-tyrosine Phe phenylalanine Trp, L-tryptophan Pgl L-phenylgiycine Pro L-proline Ile L-isoieucine Leu L-leucine, NMe norleucine Val L-valine Nal L-naphthyiaianine Hyp L-hydroxyproline Boc t. -butyrloxycarbonyi Z benzyioxycarbonyi Bpoc biphenylyipropyloxycarbonyl, Ddz dimethyridimethoxybenzyiox-ycarbonyl Fmoc fluoranymethyloxycarbonyj.

DMF dimethyiformamide HOBt 1-hydroxysuccinimide Ac acetyl, SUC succinimidyl BOP benzotriazol-1-yl-oxy-tr.is (dJmethylamino) phosphonium hexafluorophosphate TBTU 2 (1H-benzotriazol-1-yl)-1, 1,3, 3-tetramethyluranium tetrafluorophosphate 8 Osn succinimide ester TFA trifluoroacetic acid DIC diisopropylcarbodiimide S-tBu tert.-butylthio Trt trityl FAB fast atom bombardment Examples Example 1: Preparation of Fmoc-Phe(SO 3

H)

10 20 grams of L-phenylalanine were dissolved in portions in S. a mixture of 17 ml of 30 strength oleum and 20 ml of cone. sulfuric acid. The mixture was heated at 100 0 C for 1 hour and then poured into 200 ml of ice-water. The acid S" was neutralized with barium hydroxide, and the barium sulfate was filtered off. The filtrate was chromatographed on a column (Dowex 50WX2, 50-100 mesh, dimensions 230 x 32 mm) with water as eluent. Evaporation of the solvent resulted in 18.5 grams of L-parasulfophenylalanine.

e **09 7.35 grams of the amino acid were taken up in 150 ml of 10 strength sodium carbonate solution. To this was added, while stirring, a solution of 10 grams of Fmoc-OSu in 300 ml of dioxane. The mixture immediately became gellike and was stirred at roomt temperature for 2 hours. The precipitate was filtered off and the dioxane was evapor- 25 ated off. The aqueous solution was extracted 3 x with ether and acidified to pH 2 with 1 N hydrochloric acid.

Another impurity was extracted with ethyl acetate. The aqueous phase was evaporated in a rotary evaporator and the crystalline residue was dried over RSicapent in vacuo.

9- Example 2: Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Phe SO3H) -Leu-OH 0.83 gramn of Fmoc-Leu-rein (0.5 inmol) was made ready in a peptide synthesizer from A~dvanced Chemtech (Louisville, Kentucky, USA) in accordance with the manufacturer's instructionis for coupling the next amino acid. Fmoc- Phe(SO 3 H) (1.5 inmol) and 2.25 mmol of HO~t were dissolved in i5 ml of DMF and 15 ml, of DMSO, and 1.6 minol of DIC wer6 added. After one hour, the mixture was added to the Leu-resin and coupling was carried out f~or two hours.

Synthesis was then completed by standard methods. TBTU with a 3-fold excess of amino acid was used for the coupling. The coupling time was 35 minutes in each case.

The peptide-resin was treated withi 27 ml of TFA, 1.5 ml 15 of ethanedithiol and 1 g of reso7~cinol for ono hour. The peptide solution was crystallized in other, filtered off and dried. Crude yield 405 mg. 110 mg of this crude peptide were purified on an HPLC column (Shandon, RP-18, 250 x 20 mm) with a 0.1 TFA/acetonitrile gradient. The 20 peptide was isolated by freeze drying (yield 48 mg). The peptide content determined after hydrolysis was IS8

C

S.

SO S

S

56 5 C S *5

S

C

C.

a

C..

S

0

OSSC

S S S ~S S S. S S S S

S.

The amino acid composition is evident diagram and was as expected.

from the attached Amino acid analysis: Phe (SO 3

H)

Asp Glu Pro Gly Ile Leu Phe 6. 95 1.94 4.18 1.10 1.00 0.90 0.90 0.99 (1) (2) (4) Testing: mg of the peptide were dissolved in 1 ml of buffer mM Tris, 150 mM NaCl pH The peptide was tested foy- the partial thromboplastin time (PTrT) comparing with 10 a peptide with the same sequence but with TPyr in place of Phe (SioH).

PTT! test: 100 microliters of standard human plasma 100 11of buffer (see above) 100 of kaolin/pathromtin (BEHRINGftAKE AG) 2 minutes at 37 0

C

100 microliters of CaCl 2 Solution reagent 4*

S

SS* S

S.

S SO* S 0 4e S 00

S

S4S*S6 0 a.

0 a..

I'M Result: Dilution 1:4 1:8 1:16 1:.32 1:64 1:.*12 8 1 :256 20 1:512 1:1324 Coagulation times in seconds Peptide Qsf examtpl e Peptide with Tyr 147.2 105.2 111.i 80.5 89.2 71.0 81.5 65.0 72.8 57.7 63.0 52.8 56.3 47.3 52.2 44.7 46.8 41.7

B

S

0650

OS

S. S 0 S B

B.

B

a *4 0*

SB

Blank (without peptide) 38.8 The amino-acid analyses and FAB masA agreed with the expected results.

spectra in each case The following peptides are prepared correspondinglyt H-Asn--Gly-Asp-Phe-Glu-Glu-I le-Pro-Glu-Glu-Phe (S0 3 H) -Leu-

OH

H-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Phe (SO 3 H) -Leu-Gln-

OH

Ac-Asp-Phe-Glu-Glu-Ile-Pzo-Glu-Glu-Phe SUAH -Leu-OH Sar-Glu-Tyr-Glu-Glu- Ile -Pro-Glu-Glu-Phe SO 3 H) -le-OH 3.3 Ac-Asn-Aa-Asp-Pgl-Giu-Glu-Iie-Pro-Giu-Glu-Phe SO 3 H) -Leu-

OH

H-Asp-Trp-Giu-Glu-I le-Pro-Glu-Glu-Phe SO 3 H) -Leu-Gln-OH H-Asn-Gly-Asp-Pgl-Glu-Glu-Iie-Pro-Giu-Glu-Phe SO 3 H) -Leu-

OH

H-Asp-Asp-Phe-Glu-Glu-I ie-Pro-Giu-Giu-Phe SO 3 H) -Asp-OH Suc-Asp-Phe-Glu-Giu-Iie-Pro-Glu-Glu-Phe SO 3 H) -Leu-OH Suc-Asp-Pgi-Glu-Giu-I ie-Pro-Glu-Glu-Phe SO 3 H) -Leu-OH Ac-Gly-Asp-Phe-Glu-Glu-Nle-Pro-Glu-Glu-Phe -Ile-Asn-

NH

2 Ac -Gly-Asp-Tyr-Glu-Glu-Val-Pro-Glu-Giu-Phe SOH) -Leu-NH 2 Suc-Glu-Ala-Asp-Tyr-Giu-Pro-Leu-Pro-Glu-Glu-Phe SO 3 H) -Leu-

OH

Ac -Gln-Ala-Asp-Phe-Asp-Asp-Phe-Asp-Asp-Phe (SO 3 H) -Ala-NH, 15A0l s h l l l ro G u G u P e( O H D L u O 15Ac -Gly-Asp-Phe-GJlu-Glu- Ile -Pro -Glu-Gu-Phe SOH) -Leu- 4l-O HA-Gly-Asp-Phe-Glu-Glu- I e-P~o-Glu-Glu-Phe SOH) -Leu- GiOH 200 HAsn-Asp -Phel-Glu-Glu-Ile-Pro-Glu-Glu-Pg l (SOH) -Le u-

(S

3 H) -Le-OH Cys-Asn-Gly-Asp-Phe-Glu-Gu-Ile-Pro-Glu-Glu-Phe SO 3 H) -Leu- Cys-OH Cys -Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Phe (SO 3 H) -Leua .25 Glri-Cys-OH ICYs-Asn-Gly-Asp-Tyr-Glu-Glu-I le-Pro-Glu-Glu-Phe (SO 3 H) -Leu- Cys-OH lb

Claims (11)

1. A peptide of the formula I Al-J, 3-A4-A5-A6-A7-A8-A9-A-Al-A2-Al3-Al4-Al5 in whiich Al is hydrogen, cysteine, one or two alkyl groups with 1.-4 carbon atoms, an acyl group with 2-10 carbon a4tms, an acyl group with 2-10 carbon atoms and another carboxyl group, or a protective group customary in peptide chemistry, e10 A2 is a bond, Asn, Asp, Gin ot Glu, A3 s abond 9 diy or Ala, A4 is Glu or Asp, is Phe, Tyr, Trp, Pg1 (phenyiglycine) or Nal A6 (naphthyialanine), is A6 is Giu or As~p, A7 is Glu, Asp, Pro or Ala, A8 is Ile, Len, Val, Nie or Phe, A9 is Pro or HyW, doA1 s luo Ap 2 All is Giu or Asp, A12 is Phe(SQ 3 H) or Phe(POH 2 (preferably in the p position) or Pg1l(SO 3 H) Or Pgl(POAH2) -,preferably in the p position), A13 is a bond, Leu, Ile, Val or Ala, *A14 is a bond, Gln, Asn, Glu, Asp or Cys and is Cys, Cys amide, an OH group of the aipha-carboxyl group, free or esterified-vyith a lower alcohol with up to 4 carbon atoms, which can also be in the form oif a carboxamide grou,, whose hydrogens can optional- ly be replaced by alkyl groups with up to 4 carbon atoms.

2. A peptide as claimed in claim 1, in which A12 is su3fophenylalanine in the D or L form. 13 a A o BA JA A BAA jA A A A d4 'oA0'Q A A' S. eqs S OAS S U A S A Sb .g

3. A peptide as claimed in cliain 1, in which A12 is suifophenyiglycine in the D on L form.

4. A peptide as claimed in claim 1, in which Al is hydrogen, methyl, acetyl, benzoyl or succinyl.

5. A peptide as claimed *nclaim 1 with the structure H-s-l-s-h-l-G r-l-l-h SO 3 H) -Leu-QH.

6. A peptide as claimed in claim 1 with the structure H-Asn-Gly-Asp-Phe-Glu-Glu- I le-Pro-Glu-Glu-Phe (SO 3 H) -Leu- OH, 10 H-Giy-Asp-Phe-Glu-Glu- Ile-Pro-Glu-Glu-Phe SO7 -Leu-Gln- OH, Ac-Asp-Phe-Glu--Glu-Ile-Pro-Glu-Glu-Phe SO 3 H) -Leu-OH, Sar-Glu-Tyr-Glu-Glu-Ile-Pro-Glu-Glu-Phe SO 3 H) -Ile-OH, AV -Asn-Aia-Asp-Pgl-Glu-Glu- Iile-Pro-Glu-Giu-Phe SO 3 H) -Leu- OH, H-Asp-Trp-Glu-Giu-Iie-Pro-Glu-Glu-Phe SO 3 h) -Leu-Gln-OH,, H-Asn-Giy-Asp-Pgi-Glu-Giu-Ile-Pro-Glu-Giu-Phe (S0 3 H) -LeuL- OH, H-Asp-Asp-Phe-Gu-Glu-Iie-Pro-Glu-Glu-Phe (SO 2 H) -Asp-OH, 20 Suc-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Phe SO 3 H) -Lelx-OH, Suc-Asp-Pgl-Glu-Glu-Ile-.Pro-Glu-Glu-Phe (SO 3 H) -Leu-OH, Ac-Gly-Asp-Phe-G2lu-Glu-Nle-Pro-Glu-Glu-Phe SO 3 H) -Ile-As n- NH 3 Ac-Gly-Asp-Tyr-Glu-Glu-Val-Pro-Glu-Giu-Phe (SO 3 H) -LeuNH 2 25 Suc-Glu-Aia-Asp-Tyr-Glu-Pro-Leu-Pro-Glu-Giu-Phe SO 3 H) -Len- OH, Pro Ac -Gln-Ala-Asp-Phe-Asp-Asp-Phej171sp-Asp-Phe (SO3H) -Ala-NH 2 Ac-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Giu-Phe (SOH) -D-Leu- OH, Ac -Giy-Asp-Phe-Glu-Glu- Ile -Pro -Glu-Glu-D-Phe SO 3 H) -Leu- Gin-OH, H-Asn-G.ly-Asp-Phe-Glu-Giu-I le-Pro-Glu-Glu-Pgl (SO 3 H) -Leu- OH, Ac-Asp-Pgl-Glu-Glu-Ile-Pro-Glu-Glu-Pgl (SO 3 H) -Leu-OH, or Ac-Asp-Nal-Glu-Giu-Ile-Pro-Glu-Glu-Phe (SO 3 H) -Len-OH. A £06.05 A S WA A A A A SB 14

7. A process for preparing a peptide as claimed in claim 1 comprising solid- phase peptide synthesis or synthesis operating in solution.

8. A process for preparing a peptide as claimed in claim 1, which comprises the peptide chain being constructed on a polymeric support by means of repetitive coupling of protected amino acids or oligopeptides and being cleaved off therefrom.

9. A process for preparing a peptide as claimed in claim 1, which comprises constructing the peptide chain in solution using protected amino acids or protected oligopeptides, and obtaining the peptide by eliminating the protective groups.

A process for preparing a peptide of the formula I, which comprises protected amino acid derivatives or peptide segments being coupled together in S solution or on a solid phase and obtained by elimination of the protective groups and, in the case of a solid phase, by cleavage off the support resin, it being possible to carry out oxidative ring closure in the case of cysteine- containing peptides.

11. A diagnostic or therapeutic agent for use as an anticoagulant which comprises a compound as claimed in claim 1 optionally in which adjunct with suitable carriers and excipients. o DATED this 17th day of May, 1993. S. BEHRINGWERKE AKTIENGESELLSCHAFT WATERMARK PATENT TRADEMARK ATTORNEYS THE ATRIUM 290 BURWOOD ROAD HAWTHORN VICTORIA 3122 AUSTRALIA PBM/KJS/ML DOC 035 AU7125591.WPC