Peptides inhibiting the blood coagulation, their process of preparation and their use.

The present invention relates to peptides which inhibit blood clotting, processes for their preparation and their use as anticoagulants.

Anticoagulants are of great therapeutic relevance in the treatment of various diseases related to blood clotting, such as disseminated intravascular coagulation, myocardial infarction and deep vein thrombosis. Anticoagulants such as antithrombin III obtained from human plasma are currently used to treat these diseases.

More recently, a polypeptide from the leech (Hirudo medicinalis) consisting of 65 amino acids has been tested as an anticoagulant. However, the use of hirudin, as this peptide is also called, is characterized by various disadvantages. A disadvantage is the problem of the low availability of this substance. Difficulties may also arise from the relatively high molecular weight of this peptide, which potentially creates the risk of antibody formation.

These disadvantages could be avoided by developing low-molecular peptides as anticoagulants which have a high degree of homology to the C-terminal region of hirudin. Such peptides are described in the application EP-A 0 276 014, EP 0 333 356 and in a publication by JM Maraganore et al., J. Biol. Chem. 264, 8692-8698 (1989).

As can be seen from this, a peptide of the structure Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-OH is of particular interest. Very particular effectiveness could be demonstrated with a peptide whose tyrosine (Tyr) is sulfated on the phenolic group. This corresponds to the rest of the native hirudin, the tyrosine of which is sulfated at position 63.

Cyclic peptides are also of interest. In addition to the sequence just mentioned at the C- and N-terminus, these contain the amino acid cysteine, which allows cyclization in the sense of a disulfide. Instead of a disulfide bridge there can be other chemical functions, preferably a linkage via an amide bond.

By its chemical nature, a sulfated tyrosine is an ester of sulfuric acid so that the bond between the sulfur atom and the phenolic oxygen atom can be broken by hydrolysis. Such peptides have the disadvantage of reduced anticoagulant activity.

According to the prior art, attempts are therefore made to achieve sulfation on tyrosine by subsequent chemical conversion. For this purpose, the unsulfated hirudin peptides are reacted with dicyclohexylcarbodiimide and sulfuric acid in organic solvents. Sulphation can also be achieved by reacting the tyrosine-containing peptide with sulfur trioxide triethylammonium salt in pyridine or chlorosulphonic acid.

However, these reactions are characterized by the disadvantages that side reactions to phenylalanine or non-selective sulfation can take place in the presence of several tyrosine residues. This can also result in high yield losses.

The present invention was therefore based on the object of eliminating these disadvantages and of providing peptides with superior physical, chemical and physiological properties.

This object was surprisingly achieved in that the amino acid tyrosine or Tyr (SO₃H) was replaced by the amino acid Phe (SO₃H) or Phe (PO₃H₂) or Pgl (SO₃H) (Pgl means phenylglycine) or Pgl (PO₃H₂) in the peptides. The sulfonate or phosphate group is preferably bonded in the para position in the phenyl ring of phenylalanine, but binding in the meta position is also possible.

The invention therefore relates to a peptide of the general formula: A1-A2-A3-A4-A5-A6-A7-A8-A9-A10-A11-A12-A13-A14-A15 wherein<dl id="dl0001"><dt>A1</dt><dd>Hydrogen, cysteine, 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 a further carboxyl group or a protective group customary in peptide chemistry,</dd><dt>A2</dt><dd>a bond, Asn, Asp, Gln or Glu,</dd><dt>A3</dt><dd>a bond, Gly or Ala,</dd><dt>A4</dt><dd>Glu or Asp,</dd><dt>A5</dt><dd>Phe, Tyr, Trp, Pgl (phenylglycine) or Nal (naphthylalanine),</dd><dt>A6</dt><dd>Glu or Asp,</dd><dt>A7</dt><dd>Glu, Asp, Pro or Ala,</dd><dt>A8</dt><dd>Ile, Leu, Val, Nle or Phe,</dd><dt>A9</dt><dd>Pro or hyp,</dd><dt>A10</dt><dd>Glu or Asp,</dd><dt>A11</dt><dd>Glu or Asp,</dd><dt>A12</dt><dd>Phe (SO₃H) or Phe (PO₃H₂) (preferably in the p-position) or Pgl (So₃H) or Pgl (PO₃H₂) (preferably in the p-position),</dd><dt>A13</dt><dd>a bond, Leu, Ile, Val, or Ala,</dd><dt>A14</dt><dd>a bond, Gln, Asn, Glu, Asp or Cys and</dd><dt>A15</dt><dd>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 present as a carboxamide function, the hydrogens of which are optionally replaced with alkyl groups with up to 4 C-atoms could be,</dd></dl> are.

If A1 is the amino acid cysteine, the amino group can also be acetylated.

The peptides according to the invention are synthesized by methods which are known per se (G. Barany and RB 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, which are in the D-, L-, or D, L-, preferably in the L-form. Sulfuric acid, which preferably also contains sulfur trioxide, is suitable as the sulfonating agent.

These aminosulfonic acids were obtained according to CD Chang et al., Int. J. Peptide Protein Res. 15, 59 (1980) known processes on the amino group with a protective group in order to be able to build up a peptide according to the invention. The following may be considered as protecting groups: Boc, Z, Bpoc, Ddz and preferably the Fmoc group.

The peptides were built up either according to a peptide synthesis method working in solution, including known procedures (E. Wünsch in "Houben-Weyl, synthesis of peptides I", G. Thieme Verlag, Stuttgart (1974)) or according to known methods (see above).

Solid phase methods, preferably using Fmoc chemistry.

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

In solid phase peptide synthesis, the peptide chain was built up on cross-linked polystyrene (1% divinylbenzene) (later called resin). Anchors based on alkoxybenzyl alcohol were used for the synthesis of peptides with free carboxyl groups. Amide anchors were used for peptide amides (Int. J. Peptide Protein Res. 34, 262-267, 1989), which produce nonalkylated peptide amides. The individual protected amino acids were installed according to a repetitive pattern:<ul id="ul0001" list-style="dash"><li>Presentation of the Fmoc-amino acid (or amid anchor) resin in a fully automated peptide synthesizer</li><li>Washing the resin with DMF, dichloromethane or N-methylpyrrolidone (approx. 15 ml / mg)</li><li>Cleavage of the Fmoc group with 20% piperidine in dimethylformamide or N-methylpyrrolidone (preferably 1 x 3 min and 1 x 10 min)</li><li>Washing out the piperidine with DMF, dichloromethane, N-methylpyrrolidone or an alcohol, preferably isopropanol</li><li>Coupling of the amino acid with a carbodiimide, preferably diisopropylcarbodiimide, optionally with the addition of HOBt, HOSu or using BOP or TBTU, optionally with the addition of HOBt, preferably in DMF or in N-methylpyrrolidone.</li></ul>

The side chains of the trifunctional amino acids were protected as follows:<ul id="ul0002" list-style="dash"><li>Asp and Glu as t-butyl ester</li><li>Hyp and Tyr as t-butyl ether</li><li>Cys as trityl ether or tert-butyl disulfide.</li></ul>

Instead of the Fmoc chemistry shown above, these peptides can also be built up using the Boc strategy (JM Stewart and JD Young "Solid Phase Peptide Synthesis" Pierce Chemical Co., 1984, pp. 71-95) because of the repetitive use the sulfonate group is not damaged by trifluoroacetic acid.

In Fmoc chemistry, the peptides were cleaved from the resin using trifluoroacetic acid, preferably with the addition of a scavenger, and crystallized with ether. After purification using reversed-phase chromatography, their composition was confirmed using amino acid analysis and FAB mass spectrometry.

If peptides containing cysteine ​​were produced, in the case of Cys (Trt) the trityl protection was removed simultaneously during the peptide cleavage, provided the cleavage mixture contained a thiol, preferably ethanedithiol, as an additive.

If Cys (StBu) was used, the S-tBu group was preferably removed after cleavage of the peptide. Known methods, such as treatment with tri-n-butylphosphine or dithiothreitol, were used for this purpose. Dithiothreitol deprotection was preferably used.

The cyclization via the SS bridge could be carried out using known oxidative processes.

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

Other cyclization processes, such as oxidation with iodine, for example in acetic acid, or K₃ [Fe (CN) ₆] are also suitable for this.

As an alternative, production by a solution-based method is possible, in which individual fragments of the entire peptide are first produced. The condensation of individual, protected amino acids to form peptide segments was carried out in solvents such as DMF and tetrahydrofuran or mixtures thereof. The coupling of the amino acids was carried out using carbodiimides, as in solid phase synthesis. Individual segments were then assembled into the entire peptides. After the protective groups had been split off, the peptides were also purified and characterized.

The peptides were tested for their effectiveness in a functional test. The following peptides were produced in detail, but the content of the invention is not restricted to them:

Abbreviations:

<dl id="dl0002"><dt>Asn</dt><dd>L-asparagine</dd><dt>Asp</dt><dd>L-aspartic acid</dd><dt>Cys</dt><dd>L-cysteine</dd><dt>Gln</dt><dd>L-glutamine</dd><dt>Glu</dt><dd>L-glutamic acid</dd><dt>Gly</dt><dd>Glycine</dd><dt>Ala</dt><dd>L-alanine</dd><dt>Tyr</dt><dd>L-tyrosine</dd><dt>Phe</dt><dd>Phenylalanine</dd><dt>Trp</dt><dd>L-tryptophan</dd><dt>Pgl</dt><dd>L-phenylglycine</dd><dt>Per</dt><dd>L-proline</dd><dt>Ile</dt><dd>L-isoleucine</dd><dt>Leu</dt><dd>L-leucine</dd><dt>Nle</dt><dd>Norleucine</dd><dt>Val</dt><dd>L-valine</dd><dt>Nal</dt><dd>L-naphthylalanine</dd><dt>Hyp</dt><dd>L-hydroxyproline</dd><dt>Boc</dt><dd>t-butyloxycarbonyl</dd><dt>Z.</dt><dd>Benzyloxycarbonyl</dd><dt>Bpoc</dt><dd>Biphenylylpropyloxycarbonyl</dd><dt>Ddz</dt><dd>Dimethyldimethoxybenzyloxycarbonyl</dd><dt>Fmoc</dt><dd>Fluorenylmethyloxycarbonyl</dd><dt>DMF</dt><dd>Dimethylformamide</dd><dt>HOBt</dt><dd>1-hydroxysuccinimide</dd><dt>Ac</dt><dd>Acetyl</dd><dt>Suc</dt><dd>Succinimidyl</dd><dt>BOP</dt><dd>Benzotriazol-1-yl-oxy-tris (dimethylamino) phosphonium hexafluorophosphate</dd><dt>TBTU</dt><dd>2 (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium tetrafluorophosphate</dd><dt>Osn</dt><dd>Succinimide ester</dd><dt>TFA</dt><dd>Trifluoroacetic acid</dd><dt>DIC</dt><dd>Diisopropyl carbodiimide</dd><dt>S-tBu</dt><dd>Tert-butylthio</dd><dt>Trt</dt><dd>Trityl</dd><dt>FAB</dt><dd>Fast atom bombing</dd></dl>

Examples

Example 1:

Production of Fmoc-Phe (SO₃H)

20 Grams of L-phenylalanine were in a mixture of 17 ml of 30% oleum and 20 ml of conc. Dissolved sulfuric acid in portions. The mixture was heated to 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, dimension 230 x 32 mm) with water as the eluent. After evaporation of the solvent, 18.5 grams of L-para-sulfophenylalanine were obtained.

7.35 grams of the amino acid were taken up in 150 ml of 10% sodium carbonate solution. A solution of 10 grams of Fmoc-OSu in 300 ml of dioxane was added with stirring. The mixture soon became gelatinous and was stirred for 2 hours at room temperature. The precipitate was filtered off and the dioxane was evaporated. The aqueous solution was extracted 3 × with ether and acidified to pH 2 with 1N hydrochloric acid.

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

Example 2:

Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Phe (SO₃H) -Leu-OH

0.83 grams of Fmoc-Leu resin (0.5 mmol) were prepared on a peptide synthesizer from Advanced Chemtech (Louisville, Kentucky, USA) according to the manufacturer's instructions for coupling the next amino acid. Fmoc-Phe (SO₃H) (1.5 mmol) and 2.25 mmol HOBt were dissolved in 15 ml DMF and 15 ml DMSO and mixed with 1.6 mmol DIC. After one hour, the batch was added to the Leu resin and coupled for two hours. The synthesis was then completed using standard procedures. For the coupling, TBTU with 3-fold amino acid excess was used. The coupling time was 35 minutes each. The peptide resin was treated with 27 ml TFA, 1.5 ml ethanedithiol and 1 g resorcinol for 1 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 gradient of 0.1 TFA / acetonitrile. The peptide was obtained by lyophilization (yield 48 mg). After hydrolysis, the peptide content was determined to be 78%.

The amino acid composition can be seen in the attached diagram and corresponded to the expected value.

Amino acid analysis:

<dl id="dl0003"><dt>Phe (SO₃H)</dt><dd>0.95 (1)</dd><dt>Asp</dt><dd>1.94 (2)</dd><dt>Glu</dt><dd>4.18 (4)</dd><dt>Per</dt><dd>1.10 (1)</dd><dt>Gly</dt><dd>1.00 (1)</dd><dt>Ile</dt><dd>0.90 (1)</dd><dt>Leu</dt><dd>0.90 (1)</dd><dt>Phe</dt><dd>0.99 (1)</dd></dl>

Testing:

10th mg of the peptide was dissolved in 1 ml of buffer (20 mM Tris, 150 mM NaCl pH 7.5). The peptide was tested in the partial thromboplastin time (PTT) against a peptide with the same sequence which, however, contained Tyr instead of Phe (SO₃H).

PTT test:

100 Microliter standard human plasma 100 microliter buffer (see above) 100 microliters of kaolin / pathromtin reagent (BEHRINGWERKE AG) 2 minutes at 37 ° C 100 microliter CaCl₂ solution

Result:

<tables id="tabl0001" num="0001"><img file="EP0443598A2_D0001.tif" /></tables>

The respective amino acid analyzes and FAB mass spectra agreed with the expected results.

The following peptides are produced accordingly:<img file="EP0443598A2_D0002.tif" />