IE910597A1 - 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 thereofInfo
- Publication number
- IE910597A1 IE910597A1 IE059791A IE59791A IE910597A1 IE 910597 A1 IE910597 A1 IE 910597A1 IE 059791 A IE059791 A IE 059791A IE 59791 A IE59791 A IE 59791A IE 910597 A1 IE910597 A1 IE 910597A1
- Authority
- IE
- Ireland
- Prior art keywords
- glu
- phe
- asp
- pro
- peptide
- Prior art date
Links
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- ALQIUGWFHKQQHV-QMMMGPOBSA-N (2s)-2-amino-3-(4-sulfophenyl)propanoic acid Chemical compound OC(=O)[C@@H](N)CC1=CC=C(S(O)(=O)=O)C=C1 ALQIUGWFHKQQHV-QMMMGPOBSA-N 0.000 description 1
- KEQGZUUPPQEDPF-UHFFFAOYSA-N 1,3-dichloro-5,5-dimethylimidazolidine-2,4-dione Chemical compound CC1(C)N(Cl)C(=O)N(Cl)C1=O KEQGZUUPPQEDPF-UHFFFAOYSA-N 0.000 description 1
- BDNKZNFMNDZQMI-UHFFFAOYSA-N 1,3-diisopropylcarbodiimide Chemical compound CC(C)N=C=NC(C)C BDNKZNFMNDZQMI-UHFFFAOYSA-N 0.000 description 1
- 239000005995 Aluminium silicate Substances 0.000 description 1
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 1
- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 description 1
- 102000004411 Antithrombin III Human genes 0.000 description 1
- 108090000935 Antithrombin III Proteins 0.000 description 1
- 101100315624 Caenorhabditis elegans tyr-1 gene Proteins 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 102000001189 Cyclic Peptides Human genes 0.000 description 1
- 108010069514 Cyclic Peptides Proteins 0.000 description 1
- CKLJMWTZIZZHCS-UHFFFAOYSA-N D-OH-Asp Natural products OC(=O)C(N)CC(O)=O CKLJMWTZIZZHCS-UHFFFAOYSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-UHFFFAOYSA-N D-alpha-Ala Natural products CC([NH3+])C([O-])=O QNAYBMKLOCPYGJ-UHFFFAOYSA-N 0.000 description 1
- 206010051055 Deep vein thrombosis Diseases 0.000 description 1
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 241000545744 Hirudinea Species 0.000 description 1
- 241000237902 Hirudo medicinalis Species 0.000 description 1
- QNAYBMKLOCPYGJ-UWTATZPHSA-N L-Alanine Natural products C[C@@H](N)C(O)=O QNAYBMKLOCPYGJ-UWTATZPHSA-N 0.000 description 1
- CKLJMWTZIZZHCS-UWTATZPHSA-N L-Aspartic acid Natural products OC(=O)[C@H](N)CC(O)=O CKLJMWTZIZZHCS-UWTATZPHSA-N 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- 239000004201 L-cysteine Substances 0.000 description 1
- 235000013878 L-cysteine Nutrition 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- 229930182816 L-glutamine Natural products 0.000 description 1
- 229930182844 L-isoleucine Natural products 0.000 description 1
- 229930182821 L-proline Natural products 0.000 description 1
- 125000000174 L-prolyl group Chemical group [H]N1C([H])([H])C([H])([H])C([H])([H])[C@@]1([H])C(*)=O 0.000 description 1
- 125000000510 L-tryptophano group Chemical group [H]C1=C([H])C([H])=C2N([H])C([H])=C(C([H])([H])[C@@]([H])(C(O[H])=O)N([H])[*])C2=C1[H] 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 102000002262 Thromboplastin Human genes 0.000 description 1
- 108010000499 Thromboplastin Proteins 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 206010047249 Venous thrombosis Diseases 0.000 description 1
- XXFXTBNFFMQVKJ-UHFFFAOYSA-N [diphenyl(trityloxy)methyl]benzene Chemical compound C=1C=CC=CC=1C(C=1C=CC=CC=1)(C=1C=CC=CC=1)OC(C=1C=CC=CC=1)(C=1C=CC=CC=1)C1=CC=CC=C1 XXFXTBNFFMQVKJ-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 229960003767 alanine Drugs 0.000 description 1
- 235000012211 aluminium silicate Nutrition 0.000 description 1
- 235000012538 ammonium bicarbonate Nutrition 0.000 description 1
- 239000001099 ammonium carbonate Substances 0.000 description 1
- 229960005348 antithrombin iii Drugs 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 229960005261 aspartic acid Drugs 0.000 description 1
- RQPZNWPYLFFXCP-UHFFFAOYSA-L barium dihydroxide Chemical compound [OH-].[OH-].[Ba+2] RQPZNWPYLFFXCP-UHFFFAOYSA-L 0.000 description 1
- 229910001863 barium hydroxide Inorganic materials 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 235000011148 calcium chloride Nutrition 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- XTHPWXDJESJLNJ-UHFFFAOYSA-N chlorosulfonic acid Substances OS(Cl)(=O)=O XTHPWXDJESJLNJ-UHFFFAOYSA-N 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 238000010511 deprotection reaction Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- BGRWYRAHAFMIBJ-UHFFFAOYSA-N diisopropylcarbodiimide Natural products CC(C)NC(=O)NC(C)C BGRWYRAHAFMIBJ-UHFFFAOYSA-N 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- UXGNZZKBCMGWAZ-UHFFFAOYSA-N dimethylformamide dmf Chemical compound CN(C)C=O.CN(C)C=O UXGNZZKBCMGWAZ-UHFFFAOYSA-N 0.000 description 1
- 208000009190 disseminated intravascular coagulation Diseases 0.000 description 1
- 150000002019 disulfides Chemical class 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 238000002825 functional assay Methods 0.000 description 1
- 229960002989 glutamic acid Drugs 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 150000003949 imides Chemical class 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- PNDPGZBMCMUPRI-UHFFFAOYSA-N iodine Chemical compound II PNDPGZBMCMUPRI-UHFFFAOYSA-N 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- NLYAJNPCOHFWQQ-UHFFFAOYSA-N kaolin Chemical compound O.O.O=[Al]O[Si](=O)O[Si](=O)O[Al]=O NLYAJNPCOHFWQQ-UHFFFAOYSA-N 0.000 description 1
- BRHPBVXVOVMTIQ-ZLELNMGESA-N l-leucine l-leucine Chemical compound CC(C)C[C@H](N)C(O)=O.CC(C)C[C@H](N)C(O)=O BRHPBVXVOVMTIQ-ZLELNMGESA-N 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 208000010125 myocardial infarction Diseases 0.000 description 1
- YYHPEVZFVMVUNJ-UHFFFAOYSA-N n,n-diethylethanamine;sulfur trioxide Chemical compound O=S(=O)=O.CCN(CC)CC YYHPEVZFVMVUNJ-UHFFFAOYSA-N 0.000 description 1
- 230000014508 negative regulation of coagulation Effects 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 229920001467 poly(styrenesulfonates) Polymers 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 229960002429 proline Drugs 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 239000002516 radical scavenger Substances 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 229960002317 succinimide Drugs 0.000 description 1
- IIACRCGMVDHOTQ-UHFFFAOYSA-N sulfamic acid Chemical class NS(O)(=O)=O IIACRCGMVDHOTQ-UHFFFAOYSA-N 0.000 description 1
- 238000006277 sulfonation reaction Methods 0.000 description 1
- 125000001273 sulfonato group Chemical group [O-]S(*)(=O)=O 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 125000004434 sulfur atom Chemical group 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- WROMPOXWARCANT-UHFFFAOYSA-N tfa trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.OC(=O)C(F)(F)F WROMPOXWARCANT-UHFFFAOYSA-N 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 150000003573 thiols Chemical class 0.000 description 1
- FGMPLJWBKKVCDB-UHFFFAOYSA-N trans-L-hydroxy-proline Natural products ON1CCCC1C(O)=O FGMPLJWBKKVCDB-UHFFFAOYSA-N 0.000 description 1
- TUQOTMZNTHZOKS-UHFFFAOYSA-N tributylphosphine Chemical compound CCCCP(CCCC)CCCC TUQOTMZNTHZOKS-UHFFFAOYSA-N 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 229960004799 tryptophan Drugs 0.000 description 1
- 150000003668 tyrosines Chemical class 0.000 description 1
- 229960004295 valine Drugs 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Diabetes (AREA)
- 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
BEHRINGWERKE AKTIENGESELLSCHAFT 90/B 007 - Ma 822 Dr. Ha/Bi Peptides which inhibit blood coagulation, processes 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 coagulation, 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 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 0 276 014, EP 0 333 356 and in a publication by J.M. Maraganore et al., J. Biol. Chem. 264, 8692-8698 (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 5 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.
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 unsulfated hirudin peptides are reacted with dicyclohexylcarbodiimide 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-select25 ive 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(SO3H) in the peptides by the amino acid Phe(S03H) or Phe(P03H2) or Pgl(SO3H) (Pgl denotes phenylglycine) or Pgl(PO3H2). 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-A11-A12-A13-A14-A15 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 customary in peptide chemistry.
A2 is a bond, Asn, Asp, Gin or Glu, 15 A3 is a bond, Gly or Ala, A4 is Glu or Asp, A5 is Phe, Tyr, Trp, Pgl (phenylglyc ine) 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, A10 is Glu or Asp, All is Glu or Asp, 25 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, 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 Peptides (E. Gross and 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 trioxide.
These amino sulfonic acids were provided, by 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 are: Boc, Z, Bpoc, Ddz and, preferably, the Fmoc group.
The peptides were synthesized either by a peptide syn20 thesis method operating in solution and entailing known procedures (E. Wiinsch 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 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 % divinyl30 benzene) (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 patterns - 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 1x3 min and 1 x 10 min) removal of the piperidine by washing with DMF, dichloromethane, N-methylpyrrolidone or an alcohol, preferably isopropanol - 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-methylpyr20 rolidone.
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 Fmoc chemistry, the peptides were eliminated from the resin using trifluoroacetic acid, preferably with the addition of a scavenger, and crystallized 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 5 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 prefer10 ably removed after elimination of the peptide. Used for this purpose were known methods, such as, for example, treatment with tri-n-butylphosphine 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 to 0.001 mM in ammonium bicarbonate buffer (0.01 M) and shaken in the air for several hours. The cyclization was followed by HPLC.
Other cyclization methods, such as, for example, oxidations with iodine, for example in acetic acid, or K3[Fe(CN)6] 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.
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 L-asparagine Asp L-aspartic acid Cys L-cysteine Gin L-glutamine Glu L-glutamic acid Gly glycine Ala L-alanine Tyr L-tyrosine Phe phenylalanine Trp L-tryptophan Pgl L-phenylglycine Pro L-proline Ile L-isoleucine Leu L-leucine Nle norleucine Val L-valine Nal L-naphthylalanine Hyp L-hydroxyproline Boc t.-butyloxycarbonyl Z benzyloxycarbony1 Bpoc biphenylylpropyloxycarbonyl Ddz dimethyldimethoxybenzyloxycarbony1 Fmoc fluorenylmethy1oxycarbony1 DMF dimethylformamide HOBt 1-hydroxysuccinimide AC acetyl Sue succinimidyl BOP benzotriazol-1-yl-oxy-tris(dimethylamino) phosphonium hexafluorophosphate TBTU 2 (ΙΗ-benzotriazol-l-yl )-1,1,3,3-tetramethyluronium tetrafluorophosphate — 8 Osn succinimide ester TFA trifluoroacetic acid DIC di i s opropylc arbodi imide S-tBu tert.-butylthio Trt trityl FAB fast atom bombardment Examples Example 1: Preparation of Fmoc-Phe(S03H) grams of L-phenylalanine were dissolved in portions in a mixture of 17 ml of 30 % strength oleum and 20 ml 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 ml of % 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 room temperature for 2 hours. The precipitate was filtered off and the dioxane was evapor25 ated off. The agueous 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 agueous phase was evaporated in a rotary evaporator and the crystalline residue was dried over RSicapent in vacuo.
Example 2: Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Phe (SO3H) -Leu-OH 0.83 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. FmocPhe(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 mmol of DIC 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 ethanedithiol and 1 g of resorcinol for one hour. The peptide solution was crystallized 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) Ile 0.90 (1) 30 Leu 0.90 (1) Phe 0.99 (1) Testing: mg of the peptide were dissolved in 1 ml of buffer (20 mM Tris, 150 mM 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 test: 100 microliters of standard human plasma 100 100 2 minutes at 37' 100 microliters of buffer (see above) of kaolin/pathromtin (BEHRINGWERKE AG) ’C of CaCl2 solution reagent Result: Dilution Coagulation times in seconds Peptide of example Peptide with Tyr 1:4 147.2 105.2 1:8 111.8 80.5 15 1:16 89.2 71.0 1:32 81.5 65.0 1:64 72.8 57.7 1:128 63.0 52.8 1:256 56.3 47.3 20 1:512 52.2 44.7 1:1024 46.8 41.7 Blank (without peptide) 38.8 The amino-acid analyses and FAB mass spectra in each case agreed with the expected results. 25 The following peptides are prepared correspondingly: H-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Phe (SO3H) -LeuOH H-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Phe (SO3H) -Leu-GlnOH .
Ac-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Phe(S03H)-Leu-OH Sar-Glu-Tyr-Glu-Glu-1 le-Pro-Glu-Glu-Phe (SO3H )-1 le-OH Ac-Asn-Ala-Asp-Pgl-Glu-Glu-Ile-Pro-Glu-Glu-Phe (SO3H) -LeuOH H-Asp-Trp-Glu-Glu-Ile-Pro-Glu-Glu-Phe (SO3H) -Leu-Gln-OH H-Asn-Gly-Asp-Pgl-Glu-Glu-Ile-Pro-Glu-Glu-Phe (SO3H) -Leu5 OH H-Asp-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Phe (SO3H) -Asp-OH Suc-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Phe (SO3H) -Leu-OH Suc-Asp-Pgl-Glu-Glu-Ile-Pro-Glu-Glu-Phe (SO3H) -Leu-OH Ac-Gly-Asp-Phe-Glu-Glu-Nle-Pro-Glu-Glu-Phe (SO3H) -Ile-Asn10 NH2 Ac-Gly-Asp-Tyr-Glu-Glu-Val-Pro-Glu-Glu-Phe (SO3H) -Leu-NH2 Suc-Glu-Ala-Asp-Tyr-Glu-Pro-Leu-Pro-Glu-Glu-Phe (SO3H) -LeuOH Ac-Gln-Ala-Asp-Phe-Asp-Asp-Phe-Asp-Asp-Phe (SO3H) -Ala-NH2 Ac-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Phe(S03H)-D-Leu-OH Ac-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-D-Phe (SO3H) -LeuGln-OH H-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Pgl (SO3H) -LeuOH Ac-Asp-Pgl-Glu-Glu-Ile-Pro-Glu-Glu-Pgl (SO3H) -Leu-OH Ac-Asp-Nal-Glu-Glu-Ile-Pro-Glu-Glu-Phe (SO3H) -Leu-OH Cys-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Phe (SO3H) -LeuCys-OH Cys-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Phe (SO3H) -Leu25 Gln-Cys-OH Cys-Asn-Gly-Asp-Tyr-Glu-Glu-Ile-Pro-Glu-Glu-Phe (SO3H) -LeuCys-OH 1. A peptide of the formula I
Claims (15)
1. Patent claims: A1-A2-A3-A4-A5-A6-A7-A8-A9-A10-A11-A12-A13-A14-A15 in which 5 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, 10 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 (phenylglycine) (naphthylalanine), or Nal 15 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, 20 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 H) or Pgl(PO 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 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.
2. A peptide as claimed in claim 1, in which A12 is sulfophenylalanine in the D or L form.
3. A peptide as claimed in claim 1, in which A12 is sulfophenylglycine 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. 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 in claim 1 with the structure H-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Phe (SO 3 H) -LeuOH, 10 H-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Phe(S0 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) -Ile-OH, Ac-Asn-Ala-Asp-Pgl-Glu-Glu-Ile-Pro-Glu-Glu-Phe (SO 3 H) -Leu15 OH, H-Asp-Trp-Glu-Glu-Ile-Pro-Glu-Glu-Phe (SO 3 H) -Leu-Gln-OH, H-Asn-Gly-Asp-Pgl-Glu-Glu-Ile-Pro-Glu-Glu-Phe(S0 3 H) -LeuOH, H-Asp-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Phe(S0 3 H) -Asp-OH, 20 Sue-Asp-Phe-Glu-Glu-1 le-Pro-Glu-Glu-Phe(S0 3 H)-Leu-OH, Sue -Asp-Pgl-Glu-Glu-1 le-Pro-Glu-Glu-Phe (SO 3 H) -Leu-OH, Ac-Gly-Asp-Phe-Glu-Glu-Nle-Pro-Glu-Glu-Phe (SO 3 H) -Ile-AsnNH 2 , Ac-Gly-Asp-Tyr-Glu-Glu-Val-Pro-Glu-Glu-Phe (SO 3 H) -LeuNH 2 , 25 Suc-Glu-Ala-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-Glu-Glu-Ile-Pro-Glu-Glu-Phe (SO 3 H) -D-LeuOH, 30 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(S0 3 H)-LeuOH, Ac-Asp-Pgl-Glu-Glu-Ile-Pro-Glu-Glu-Pgl (SO 3 H) -Leu-OH, or 35 Ac-Asp-Nal-Glu-Glu-!le-Pro-Glu-Glu-Phe(S0 3 H) -Leu-OH. Ί.
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 the 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 the 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 the 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 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 or therapeutic agent according to claim 11, substantially as hereinbefore described.
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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 IE66493B1 (en) | 1996-01-10 |
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EP (1) | EP0443598B1 (en) |
JP (1) | JPH0770181A (en) |
KR (1) | KR910021412A (en) |
AT (1) | ATE122684T1 (en) |
AU (1) | AU640739B2 (en) |
CA (1) | CA2036815A1 (en) |
DE (2) | DE4005591A1 (en) |
DK (1) | DK0443598T3 (en) |
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CA2064231A1 (en) * | 1989-07-20 | 1991-01-21 | John M. Maraganore | Combinations and methods for treating or preventing thrombotic diseases |
WO1991002348A1 (en) * | 1989-08-07 | 1991-02-21 | Motorola, Inc. | Speech recognition using spectral line frequencies |
US5196404B1 (en) * | 1989-08-18 | 1996-09-10 | Biogen Inc | Inhibitors of thrombin |
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1990
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1991
- 1991-02-20 KR KR1019910002692A patent/KR910021412A/en not_active Application Discontinuation
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- 1991-02-22 DK DK91102607.8T patent/DK0443598T3/en active
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EP0443598A2 (en) | 1991-08-28 |
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AU7125591A (en) | 1991-08-29 |
KR910021412A (en) | 1991-12-20 |
CA2036815A1 (en) | 1991-08-23 |
EP0443598A3 (en) | 1992-01-08 |
EP0443598B1 (en) | 1995-05-17 |
JPH0770181A (en) | 1995-03-14 |
PT96836A (en) | 1991-10-31 |
PT96836B (en) | 1998-07-31 |
DE59105479D1 (en) | 1995-06-22 |
AU640739B2 (en) | 1993-09-02 |
DK0443598T3 (en) | 1995-09-25 |
DE4005591A1 (en) | 1991-09-05 |
ES2073601T3 (en) | 1995-08-16 |
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