IE66493B1 - Peptides which inhibit blood coagulation processes for the preparation thereof and the use thereof - Google Patents

Peptides which inhibit blood coagulation processes for the preparation thereof and the use thereof

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Publication number
IE66493B1
IE66493B1 IE59791A IE59791A IE66493B1 IE 66493 B1 IE66493 B1 IE 66493B1 IE 59791 A IE59791 A IE 59791A IE 59791 A IE59791 A IE 59791A IE 66493 B1 IE66493 B1 IE 66493B1
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glu
asp
phe
pro
peptide
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IE59791A
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IE910597A1 (en
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Werner Stueber
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Behringwerke Ag
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/815—Protease inhibitors from leeches, e.g. hirudin, eglin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
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  • General Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Hematology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

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

BEBRIHGHEKKS jmrc3HGBSEXI£CBaFT 90/B 007 - Ma 822 Dr- Ba/Bi Peptides which inhibit: blood coacnalation,, processes. ..fox the preparation thereof and the use thereof Th® present invention relates to peptides which inhibit blood coagulation, processes for th© preparation thereof and the wee thereof as anticoagulants.
Anticoagulants are of great therapeutic relevance in the treatment of various disorders affecting blood coagu10 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 HI which is obtained from 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.
It has been possible to circumvent these disadvantages by developing low molecular weight peptides as anticoagulants, which have a high degree of homology with the Cterminal region of hirudin. Peptides of this type are described in the application EP-A 0 27S 014, EP 0 333 356 and in a publication by j.m. Maraganore et al-, J. Biol.
Chem. 264, 8692-8693 (1989).
It is evident therefrom that a peptide of the structure Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-OH is of particular Interest. A peptide whose tyrosine (Tyr) is 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 cyclisation in the form of a disulfide. A disulfide bridge can also be replaced by other chemical functions, preferably a connection via an amide linkage.
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. j?or this purpose, the unsulfated hirudin peptides are reacted with dicycloheaylcarbodiimide and sulfuric acid In organic solvents. 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 can 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(SOaH) in the peptides by the amino acid PhefSOjH) or j?he(PCyj2} or Pgl(SOaB) (Pgl denotes phenylglycine) or Pgl(PO3H2)’ 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 ox the formulas A1-A2-A3~A4-A5-AS-A7-A8-A9~A1Q-A11-A12-AI3-A14-A15 In which Al Is hydrogen, cysteine, one or two alkyl groups with 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 customary in peptide chemistry, A2 is a bond, Asn, Asp, Gin or Glu, A3 is a bond, Gly or Ala, A4 is Glu or Asp, A5 is Phe, Tyr, Trp, Pgl (phenyl glycine) or Nal (naphthylalanine), AS Is Glu or Asp, A7 Is Glu, Asp, Pro or Ala, A8 is Xie, Leu, Val, Nle or Phe, A9 is Pro or Hyp, A10 Is Glu or Asp, All Is Glu or Asp, A12 Is Ph®(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, lie, Val or Ala, A14 is a bond. Gin, 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 carbon atoms, which can also be in the form of a carboxamide group whose hydrogens can optionally be replaced by alkyl groups with up to 4 carbon atoms.
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 (G. Barany and R.B. Merrifield in "The Peptides9* I. Meienhofer, Ed.))» The amino acids Phe(SO3H) or Pgl(SO3H) 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 trloxide.
These amino sulfonic acids were provided, fey methods known from C. D. Chang et al,.. , Int. J. Peptide Protein Res. 15, 59 (1980), with a protective group on the amino group in order to be able to synthesize a peptide according to the invention. Suitable protective groups in this context ares Boc, Bpoc, Ddz and, preferably, the Pmoc groupThe peptides were synthesized either by a peptide synthesis method operating in solution and entailing known procedures (B. WQnsch In Bouben-fteyl, Synthese won Peptiden I (Synthesis of Peptides), G. Thieme Verlag, Stuttgart (1974)) or by solid-phase methods which are likewise known (see above), in which case Pmoc chemistry was preferably used.
The sulfonate or phosphate group was employed unprotected in the synthesis.
In the solid-phase peptide 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 alkoxybenzvl alcohol. Amide anchor® (Int. jr„ Peptide Protein Res. 34, 262-267, 1989) which produce 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 imoc-amino acid (or .amide anchor)-resin into a completely automatic peptide synthesiser - washing of the resin with DMF, dichloromethane or Nmethylpyrrolldone (about 15 ml/mg) elimination of the B'moc group with 20 % piperidine in dimethylfoxmamide or N-methylpyrrolidone (preferably 1 x 3 min and 1 x 10 rain) removal of the piperidine by washing with EMMF, dichloromethane, N-methylpyrrolidone or an alcohol, preferably isopropanol - coupling of the .amino acid using a carbodiimide, preferably diisopropylcurbodiimide, where appropriate with the addition of HOBt, BOSu or with the us© of BOP or ΤΒΤΉ, where appropriate with the addition of HOBt, preferably in DMF or In N-methylpyrrolidone.
The sidechains of the trifunctional amino acids were protected as follows: - Asp and Glu as t.-butyl ester - Hyp and Tyr as t.-butyl ether - Cys as trityl ether or tert.-butyl disulfide.
In place of the Fmoc chemistry described above, these peptides can also be synthesized using the Boc strategy (J.M- 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 Baoc chemistry, the peptides were eliminated from the resin using trifluoroacetic acid, preferably with the addition of a scavenger, and crystallized - 5 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) «as employed, the S-tBu group was preferably removed after elimination of the peptide. Used for this purpose were known methods, such as, for example, treatment with tri-n-butylphoephine or dithiothreitol. Dithiothreitol deprotection was preferably used.
The cyclization via S-S bridges could be carried out by known oxidative methods.
The cysteine-containing peptide was preferably dissolved in a concentration of 0.1 fo 0.001 mH in ammonium bicarbonate buffer (0.01 H) and shaken in the air for several hours. The cyclization was followed by HPLC.
Other cyclisation methods, such as, for example, oxidations with iodine, for example in acetic acid, or X3[Fe(OT)63 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 DM? 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.
The peptides were tested for their activity in a functional assay.
The following specific peptides were prepared, but the contents of the invention are not confined to them: Abbreviations; Asn Asp Cys Gin L-asparagine L-aspartic acid L-cysteine L-glutamine 10 Glu L-glutamic acid Gly glycine Ala L-alanine Tyr L-tyrosIne Phe phenylalanine 15 Trp L-tryptophan Pgl L-phenylglycine Pro L-proline II® L-isoleucine Leu L-leucine 20 Nle norleucine Val L-valin©’ Mai L-naphthy1alanine Hyp L-hydroxyproline Boc t.-butyloxycarbonyl 25 s bensyloxyc arbony1 Bpoc biphenylylpropyloxycarbonyl Ddz dimethyldimethoxybenssyloxycarhonyl Fmoc fluorenylmethyloxycarbony1 DMF dimethyl f ormasaide 30 EOBt 1 -hydroxysucc inixaide AC acetyl Sue succinimidyl BOP bensotriasol-l~yl-oxy~tris(dimethylamino) phosphonium hexafluorophosphate 35 TBTU 2(IH-bensotriazol-l-yl)-1,1,3,3-tetramethyluroniuxa tetrafluorophosphate Osn TFA DIC S-tBu Trt FAB succinimide ester trifluoroacetic acid di i s opropy1c arbodi imide tert.-butylthio trityl fast atom bombardment Examples Example Is Preparation of Pmoc»Phe(SQ3H) grams of L-phenylalanine were dissolved In portions in a mixture of 17 ml of 30 % strength oleum and 20 sal of cone» sulfuric acid. The mixture was heated at 100°C for 1 hour and then poured into 200 ml of ice-water. The acid 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. 7.35 grams of the amino acid were taken up In 150 »1 of 10 % strength sodium carbonate solution. To this was added, while stirring, a solution of 10 grams of BSaoc-OSu in 300 ml of dioxane. The mixture immediately became gellike and was stirred rat room temperature for 2 hours. Th© precipitate was filtered off and the dioxane was evaporated 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 th© crystalline residue was dried over aSIoapent in vacuo.
Example 2: Asn~Gly-Asp-Phe-Glu~Glu-Ile-Pro-Glu~Glu-Phe (SO3H) -Leu-OH 083 gram of Fmoc-Leu-resin (0.5 mmol) was made ready in a peptide synthesizer from Advanced Chemtech (Louisville, Kentucky, USA) in accordance with the manufacturer's instructions for coupling the next ,amino acid. HmocPhe(SO3H) (1.5 mmol) and 2.25 mmol of HOBt were dissolved in 15 ml of DMF and 15 ml of DMSO, and 1.6 sraaol of DIG were added. After one hour, the mixture was added to the Leu-resin and coupling was carried out for 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 with 27 ml of TFA, 1.5 ml of eth&nedithxol and 1 g of resorcinol for one hour. The peptide solution was crystallised In ether, 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 peptide was isolated by freeze drying (yield 48 mg). The peptide content determined after hydrolysis was 78 %.
The amino acid composition is evident from the attached diagram and was as expected.
Amino acid analysis: Phe(SO3H) 0.95 (1) 25 Asp 1.94 (2) Glu 4.18 (4) Pro 1.10 (1) Gly 1.00 (1) He 0.90 (1) 30 Leu 0.90 (1) Phe 0.99 (I) Testing; mg of the peptide were dissolved in 1 ml of buffer (20 rnM Tris, 150 mSi NaCl pH 7.5). The peptide was tested for the partial thromboplastin time (PTT) comparing with a peptide with the same sequence but with 'Tyr in place of Phe(S03H).
PTT tests 100 microliters of standard human, plasma 100 of buffer (see above) 100 of kaolin/pathromtin reagent 2 minutes at 37 (BEHRING^RKS AG) ®C 100 micro1iters of CaCl2 solution Result: Dilution Coagulation times in seconds Peptide of example Peptide with Tyr 1:4 147.2 105.2 1 = 8 111.8 80.5 1=16 89.2 71.0 1:32 81.5 65.0 1 = 64 72.8 57.7 1 = 128 53.0 52.8 1:255 56.3 47.3 1 = 512 52.2 44.7 1=1024 46.3 41.7 Blank (without peptide) 38.8 The amino-acid analyses and FAB mass spectra in each case agreed with the expected results.
The following peptides are prepared correspondingly: sa-Asn-Giy-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Gls-Phe (SOsB) «LeuOH H-Gly-Asp-Phe-Glu-Glu-Ile-Fro-Glu-Glu-Phe (SQ3H) -Leu-Gln0Ξ Ac-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Phe (SOgB) -Leu-QH Sar-Glu-Tyr-Glu-Glu-IIe-Pro-Glu-Glu-Phe (SOgH) -Ile-QH Ac-Asn-Ala-Asp-Pgl-Glu-Glu-Ile-Pro-Glu-Glu-Phe (SO3H) -LeuOB H-Asp-Trp-Glu-Glu-Ile-Pro-Glu-Glu-Phe (SO3H) -Leu-Gln-QH H-Asn-Gly-Asp-Pgl-Glu-Glu-Ils-Pro-Glu-Glu-Ph© (SOsH) -LouOH H-Asp-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Phe (SO3H) -Asp-OH Suc-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Phe (SO3B) -Leu-OH Suc-Asp-Pgl-Glu-Glu-Ile-Pro-Glu-Glu-Phe (SO3H) -Leu-OH Ac-Gly-Asp-Phe-Glu-Glu~Nle~?ro-Glu-Glu-Phe (SO3H) -Ile-Asnhh2 Ac-Gly-Asp-Tyr-Glu-Glu-Val-Pxo-Glu-Gl«-Phe (SO3H) -Leu-NHs Suc-Glu-Ala-Asp-Tyr-Glu-Pi»-Leu-Pxo-Glu-Glu-Phe (SO3H) -LeuOH Ac-Gln-Ala-Asp-Phe-Asp-Asp-Phe-Asp-Asp-Phe (SO3H) -Ala-HHs Ac-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Phe (SO3H) -D-Leu-OH Ac-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-D-Phe (SO3Bt) -LeuGln-OH H-Asn-Gly-Asp-Pfoe-Glu-Glu-Ile-Pro-Glu-Glu-Pgl (SO3H) -LeuOH Ac-Asp-Pgl-Glu-Glu-Ile-Pro-Glu-Glu-Pgl (SO3B) -Leu-OH Ac-Asp-Nal-Glu-Glu-Ile-Pro-Glu-Glu-Phe (S03H) -Leu-OH Cys-Asn-Gly-Asp-Phe-Glu-Glu-Xle-Pxo-Glu-Glu-Phe (SO3H) -LeuCys-OH Cys-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pxo-Glu-Glu-Phe (SO3H) -LeuGlxx-Cys-OH Cys-Asn-Gly-Asp-Tsx-Glu-Glu-Ile-Pxo-Glu-Glu-Phe (SO3H) -LeuCys-OH HOE 90/B 007

Claims (15)

1. Patent claims s
1. A peptide of the formula I A1-A2-A3-A4-A5-A6-A7-A8-A9-A10-A11-A12-A13-A14-A15 * in which Al is hydrogen, cysteine, one or two alkyl groups with 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 customary in peptide chemistry, A2 is a bond, Asn, Asp, Gin or Glu, A3 is a bond, Gly or Ala, A4 is Glu or Asp, A5 is Ph®, Tyr, Trp, Pgl (phenyl glycine) or Nal (naphthylalanine), AS is Glu or Asp, A7 is Glu, Asp, Pro or Ala, A8 is Ils, Leu, Val, Nle or Ph®, A9 is Pro or Hyp, A10 is Glu or Asp, All is Glu. or Asp, A12 is Phe(SO 3 H) or Phe(PO 3 H 2 ) (preferably in the p position) or Pgl(SO 3 S) or Pgl(PO 3 H 2 ) (preferably in the p position), A13 is a bond. Leu, lie, Val or Ala, A.14 Is a bond. Gin, 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 carbon, atoms, which can also be in the fora of a carboxamide group whose hydrogens can optionally be replaced by alkyl groups with up to 4 carbon (atoms.
2. A peptide as claimed In claim 1, in which A12 is sulf©phenylalanine in the D or L fora.
3. A peptide as claimed in claim 1, in which A12 is sulfophenylglyeine in the D or 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 in claim 1 with the structure H-Asn-Gly-Asp-Phe-Glu-Glu-Pro-Glu-Glu-Phe (SO 3 H) -Leu-OH.
6. A peptide as claimed la claim 1 with the structure H-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Phe (SO 3 H) -LeuOH, H-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Phe (SO 3 H) -Leu-GinOH, 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) -Xle-OH, Ac-Asn-Ala-Asp-Pgl-Glu-Glu-Xle-Pro-Glu-Glu-Phe (SO 3 H) -LeuOH, H-Asp-Trp-Glu-Glu-Ile-Pro-Glu-Glu-Phe (SO 3 H) -Leu-Gln-OH, H-Asn-Gly-Asp-Pgl-Glu-Glu-Ile-?ro-Glu-Glu-?he (SO a H) -LeuOH, H-Asp-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Phe (SO 3 H) -Asp-OH, Suc-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Phe (SO 3 H) -Leu-OH, Suc-Asp-Pgl-Glu-Glu-Xie-Pro-Glu-Glu-Phe (SO 3 H) -Leu-OH, Ac-Gly-Asp-Phe-Glu-Glu-Nle~Pro-Glu-Glu-Phe (SO 3 H) -Ils-Asnbn 2 , Ac-Gly-Asp-Tyr-Glu-Glu-Val-Pro-Glu-Glu-Phe (SO 3 H) -LeuHH 2 , Suc-Glu-Aia-Asp-Tyr-Glu-Pro-Leu-Pro-Glu-Glu-Phe (SO 3 H) -LeuOH, Ac-Gln-Ala-Asp-Phe-Asp-Asp-Phe-Asp-Asp-Phe (SO 3 H) -Ala-NH 2 , Ac-Gly-Asp-Phe-GXu-Gln-Ile-Pro-Glu-Glu-Phe (SQ a H) -D-LeuOH, Ac-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-D-Phe (SO 3 H) -LeuGln-OH, H-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Pgl (SO a H) -LeuOH, Ac-Asp-Pgl-Glu-Glu-Ile-Pro-Glu-Glu-Pgl (SO 3 H) -Leu-OH, or Ac-Asp-Nal-Glu-Glu-Xle-Pro-Glu-Glu-Ph© (SO a H) -Leu-OH.
7. A process for preparing a peptide as claimed in claim 1 by solid-phase peptide synthesis or synthesis operating in solution.
8. A process for preparing a peptide as claimed in 5 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 10 claim 1, which comprises constructing th® peptide chain In solution using protected .amino acids or protected oligopeptides, and obtaining the peptide by eliminating the protective groups.
10. A process for preparing a peptide of th® formula I, 15 which comprises protected amino acid derivatives or peptide segments being coupled together in 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 20 oxidative ring closure in th® case of cysteine-containing peptides»
11. a diagnostic or therapeutic agent containing a compound as claimed In claim 1.
12. A. peptide of the formula I given and defined in S' claim 1, substantially as hereinbefore described and exemplified.
13. A process for preparing a peptide of the formula I given and defined in claim 1, substantially as hereinbefore described and exemplified.
14. A peptide of the formula I given and defined in claim 1, whenever prepared by a process claimed in a preceding claim.
15. A. diagnostic ox- therapeutic agent according to claim 11, substantially as hereinbefore described.
IE59791A 1990-02-22 1991-02-21 Peptides which inhibit blood coagulation processes for the preparation thereof and the use thereof IE66493B1 (en)

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DE4005591A DE4005591A1 (en) 1990-02-22 1990-02-22 THE HERBAL INHIBITING PEPTIDES, METHOD FOR THEIR PRODUCTION AND THEIR USE

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IE66493B1 true IE66493B1 (en) 1996-01-10

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AU640739B2 (en) 1993-09-02
PT96836A (en) 1991-10-31
AU7125591A (en) 1991-08-29
ATE122684T1 (en) 1995-06-15
DK0443598T3 (en) 1995-09-25
EP0443598A3 (en) 1992-01-08
JPH0770181A (en) 1995-03-14
ES2073601T3 (en) 1995-08-16
PT96836B (en) 1998-07-31
DE59105479D1 (en) 1995-06-22
IE910597A1 (en) 1991-08-28
EP0443598A2 (en) 1991-08-28
KR910021412A (en) 1991-12-20
CA2036815A1 (en) 1991-08-23
DE4005591A1 (en) 1991-09-05
EP0443598B1 (en) 1995-05-17

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