CH641761A5 - Tripeptide derivatives for the determination of plasminogen activators and trypsin - Google Patents

Abstract

Tripeptide derivatives of the formula R<1>-X-Y-Z-NH-R<2> in which X represents a group of the formula <IMAGE> , in which R<3> designates a straight-chain or branched alkyl radical having 1 to 7 carbon atoms or a cyclohexyl or cyclohexylmethyl radical, Y represents a seryl group or a group of the formula -NH-(CH2)n-CO-, in which n designates an integer from 1 to 7, Z represents an arginyl or lysyl group, R<1> represents hydrogen or an acyl group and R<2> represents an aromatic hydrocarbon radical optionally carrying substituents.

Description

       

  
 

** WARNING ** beginning of DESC field could overlap end of CLMS **. 

 



   PATENT CLAIMS
1.  Tripeptide derivatives of the formula R'-X-Y-Z-NH-R2 in which X is a group of the formula
EMI1. 1
 in which R3 denotes a straight-chain or branched alkyl radical having 1 to 7 carbon atoms or a cyclohexyl or cyclohexylmethyl radical, Y represents a seryl group or a group of the formula -NH- (CH2) n-CO-, in which n is an integer from 1 to 7, Z represents an arginyl or lysyl group, R 'represents hydrogen or an acyl group and R2 represents an aromatic hydrocarbon radical which may have substituents. 



   2nd  Tripeptide derivatives according to claim 1, characterized in that they with a mineral acid, for. B.  HCl, HBr, H2S04 or H3PO4, or an organic acid, e.g. B. 



  Formic, acetic, oxalic or tartaric acid are protonized. 



   3rd  Tripeptide derivatives according to claim 1, characterized in that the acyl group R 'has the partial formula R4-CO-, in which R4 a) an aliphatic hydrocarbon residue with 1 to 17 carbon atoms, optionally bearing an amino group in the o-position, b) an araliphatic hydrocarbon residue, the aliphatic radical has 1 to 17 carbon atoms and its aryl radical optionally carries an amino group, c) represents a cycloaliphatic hydrocarbon radical optionally bearing an amino or aminomethyl group, d) represents an aromatic hydrocarbon radical optionally bearing an alkyl, amino or aminoalkyl group or e) a benzyloxy group. 



   4th  Tripeptide derivatives according to claim 1, characterized in that the sulfonyl group Rl is an alkanesulfonyl group having 1 to 17 carbon atoms in the alkane radical, for. B. 



  a methane or ethanesulfonyl group, or an arylsulfonyl group optionally carrying at least one lower alkyl substituent, e.g. B.  is a benzene, p-toluene or naphthalenesulfonyl group. 



   5. Tripeptide derivatives according to claim 1, characterized in that R2 is a p-nitrophenyl, naphthyl or 4-metoxy-2-naphthyl group. 



   The invention relates to tripeptide derivatives for the determination of plasminogen activators, their inhibitors and plasminogen preactivators as well as trypsin, trypsin inhibitors and trypsinogen. 



   The human organism produces several activators which convert the plasminogen proenzyme into the active plasmin lysis enzyme.  This group of activators include e.g. B.  Tissue and blood kinase and urokinase.  These activators play an important role in the mechanism of blood coagulation.  If these activators are released abnormally large, there is a risk of increased fibrinolysis activity and thus an increased tendency to bleed or hemorrhage.  Conversely, if these activators are released too weakly, the balance between coagulation capacity and fibrinolysis is disturbed and, as a result, there is an increased risk of thrombosis.  The determination of plasminogen activators in body fluids and tissue extracts is therefore of great importance in clinical practice. B.  from I. 

  Witt in Biochemistry of Blood Coagulation and Fibrinolysis, Verlag Chemie, Weinheim, 1975, p.  119, is defined as follows: The determination of the fibrinolytic activity in plasma or serum serves firstly to identify hyperfibrinolytic states which are associated with various clinical pictures.  In addition, good results in the lysis of intravascular thrombi by administration of plasminogen activators have been achieved in recent years.  To control this thrombolytic therapy, measurements of the fibrinolytic activity are also essential.     F.  E.  . Smyrniotis et al.  [Thromb.  Diath.  et Haemorrh.  Vol.    III, 257-70 (1959)] lead u. a.  the following: The excretion of urokinase in normal people is independent of age, gender and amount of urine. 

  The urokinase excretion is increased after the occurrence of a myocardial infarction and after an attack of coronary insufficiency.  Elimination is reduced in patients with carcinosis, cardiac congestion and uremia.  These differences suggest that significant changes in the fibrinolytic system of the plasma can occur as a result of these diseases.  
So far, there are no really reliable methods for determining plasminogen activators in body fluids and organ extracts.  In principle, three methods are known. 



   1.  Spontaneous lysis of a blood clot.  Blood is allowed to clot spontaneously or by adding thrombin and the spontaneous lysis of the clot is observed at 37 ° C.     Lysis usually takes 24 hours.  This method is unspecific, since the activator effectiveness is not measured directly, but indirectly via the lysis enzyme plasmin (see I.  Witt Biochemistry of Blood Coagulation and Fibrinolysis, Verlag Chemie, Weinheim, 1975, p.  119). 



   2nd  Hydrolysis of casein.  This method uses the property of the plasmin to hydrolytically break down casein.  Casein is incubated with the sample to be examined.  At the beginning and at the end, an aliquot of the incubation mixture is mixed with trichloroacetic acid.  In the supernatant, the tyrosine content is determined by spectrophotometric measurement at 280 nm, from which the activator effectiveness can be approximately calculated indirectly [L.  F.  Remmert et al. , J.  Biol.  Chem.  181, 431 (1949)].    



   3rd  Esterolytic method (for urokinase).  This method makes use of the property of urokinase to catalyze the hydrolysis of the methyl N-acetyl-L-lysine directly.  This method has also to create the so-called.  CTA urokinase unit served. 



  (CTA = Committee on Thrombolytic Agents of the National Heart Institute, USA).  A CTA unit is the amount of urokinase that releases 46.2 x 10-3 pLM M methanol from Nt-acetyl-L-lysine methyl ester in 1 hour at 37 "C (see N. U.  Bang et al. , Thrombosis and Bleeding Disorders, Georg Thieme Verlag, Stuttgart, 1971, p.  377).  This method can only be used for the determination of pure urokinase preparations, but is not suitable for the determination of urokinase in body fluids or tissue extracts, since the ester mentioned contains other proteolytic enzymes (e.g. B.  Plasmin, plasma kallikrein, etc. ) is split faster than by urokinase.   



   Attempts have been made to synthetic amide and peptide sub



  to develop strate for the determination of urokinase. 



  However, these attempts have been unsuccessful [see e.g. B. 



  W.  Troll et al. , Journal of Biological Chemistry 208, 85 (1954)].    



   In attempts to develop a synthetic substrate for the determination of kallikrein, the following tripeptide-p-nitro-anilides of the formula Pro-Phe-Arg-pNA and Val-Gly-Arg-pNA were used based on theoretical considerations
A B synthesized.  These tripeptides contain the last 3 C-terminal amino acids of the cleavage products resulting from the action of kallikrein on kininogen.  It could therefore have been expected that these two substrates would be hydrolyzed by kallikrein, but this was only true for the substrate of formula A.  However, it has surprisingly been found that the substrate of the formula B which is not at all cleavable by kallikrein is rapidly cleaved amidolytically by urokinase and other plasminogen activators. 



   Based on this knowledge, the substrates of the formula: R1-X-Y-Z-NH-R2 were synthesized, wherein X is a group of the formula
EMI2. 1
 is in which R3 represents a straight-chain or branched alkyl radical having 1 to 7 carbon atoms, Y represents a seryl group or a group of the formula -NH- (CH2) n-CO-, in which n represents an integer from 1 to 7, Z Is arginyl or lysyl, R 'represents hydrogen or an acyl group and R2 denotes an aromatic hydrocarbon radical which may have substituents. 



   The group -NH-R2 is a chromogenic group which is split off under the hydrolytic action of the enzymes and forms the cleavage product NH2-R2, the amount of which can be measured by photometric, spectrophotometric or fluorescence-photometric methods. 



   The substrates according to the invention can with a mineral acid, for. B.  HCl, HBr, H2SO4 or H3PO4, or with an organic acid, e.g. B.  Formic, acetic, oxalic or tartaric acid may be protonized. 



   R2 can be a p-nitrophenyl, naphthyl or 4-methoxy2-naphthyl group. 



   The acyl group denoted by R 'in the formula I can be characterized by the following group formula: R4-COworin R4 (a) an aliphatic hydrocarbon radical with 1 to 17 carbon atoms which optionally has an amino group in the co-position, (b) an araliphatic hydrocarbon radical, its aliphatic radical contains 1 to 17 carbon atoms and its aryl radical optionally carries an amino group, (c) a cycloaliphatic hydrocarbon radical optionally bearing an amino or aminomethyl group, (d) an aromatic hydrocarbon radical optionally bearing an alkyl, amino or aminoalkyl group or ( e) denotes a benzyloxy group. 



   R4 can in particular be an alkyl group such as methyl, ethyl, propyl, butyl, pentyl, etc. until heptadecyl. 



  These alkyl groups can carry an amino group in the o-position.  Thus R4 can e.g. B.  be a co-aminopropyl, o> -amino-pentyl or co-aminononyl group.  R4 may also be benzyl, 2-phenylethyl, 3-phenylpropyl, etc. denote up to 1 l-phenylundecyl group.  The phenyl radical of the groups mentioned can carry an amino group in the p-position.  Thus, R4 can be p-aminobenzyl, 2- (p-aminophenyl) ethyl, 3- (p-aminophenyl) propyl, etc.  be up to 1 l- (p-aminophenyl) -undecyl group.  R4 may also represent a cyclohexyl, 4-aminocyclohexyl, 4-amino-methylcyclohexyl, 4-aminoethylcyclohexyl, 4-aminopropylcyclohexyl or 4-aminobutylcyclohexyl group. 

  R4 can also represent a phenyl, ot-naphthyl, B-naphthyl or biphenyl group.  These groups can in turn carry an amino group, an aminoalkyl group or an alkyl group.  The alkyl in the aminoalkyl and alkyl groups mentioned can, for. B.  Be methyl, ethyl, propyl, isopropyl, butyl or isobutyl.  Finally, R4 can represent an alkoxy group with 1 to 12 carbon atoms and a straight or branched chain or an aralkyl group whose alkylene radical contains 1 to 4 carbon atoms. 



   The sulfonyl group denoted by R 'in formula I can be an alkanesulfonyl group whose alkane radical is straight-chain or branched and contains 1 to 12 carbon atoms, e.g. B. 



  a methanesulfonyl group, or an arylsulfonyl group, the aryl radical of which is mono- or polynuclear and can carry substituents, e.g. B.  a benzenesulfonyl, p-toluenesulfonyl or 2-naphthalenesulfonyl group. 



   The tripeptide derivatives according to the invention are suitable for the determination of plasminogen activators, e.g. B.  Urokinase, inhibitors of the same and plasminogen preactivators and of trypsin, trypsin inhibitors and trypsinogen in human and mammalian body fluids and in tissue extracts.  The determination is carried out by reacting the body fluids or tissue extracts mentioned with one of the substrates defined above and determining the amount of the NH2R2 cleavage product formed on the substrate by the hydrolytic effect of the biologically active factors mentioned by photometric, spectrophotometric or fluorescence spectrophotometric methods. 

 

   The preparation of the substrate according to the invention can be carried out according to various, e.g. T.  known methods take place:
1) In the first method, the chromogenic groups are attached to the C-terminal amino acid group.  These groups simultaneously perform the function of C-terminal carboxyl protecting groups during the step-wise linking of amino acids to the desired peptide structure.  The remaining protective groups are split off selectively from the final product without affecting the chromogenic group.  This method is e.g. B.  in Peptide Synthesis by Miklos BODANSZKY et al. , Interscience Publishers, 1966, pages 163-, 1965. 



   2) In the second method, the chromogenic group is coupled to the fully assembled peptide structure, in that after the step-by-step construction of the desired peptide structure has taken place, the C-terminal carboxyl group is first released by alkaline hydrolysis of the ester, whereupon the chromogenic group is attached to the Carboxyl group is attached.  The remaining protective groups are finally split off selectively, under conditions in which the chromogenic group remains intact.  This method is described in Peptide Synthesis, see above, pages 43 and 44. 



   To protect the N -amino groups during the step-by-step build-up of the peptide chains, conventional, known amino-protecting groups which can be cleaved off selectively can be used.  It is primarily Cbo, MeOCbO, NO2Cbo, MCbo, BOC, TFA or Formyl.  The a-carboxyl group of the amino acids can be made reactive by various known methods, e.g. B.  by preparing the p-nitrophenyl ester derivatives, trichlorophenyl ester derivatives, pentachlorophenyl ester derivatives, N-hydroxysuccinimide ester derivatives and isolating these derivatives or by preparing in situ the acid azides or acid anhydrides, which may be either symmetrical or asymmetrical. 



   The carboxyl group can also be activated by means of a carbodiimide, such as N, N'-dicyclohexylcarbodiimide. 



   The carboxyl group, which is C-terminal in the peptide derivatives, is protected either by means of the chromophoric amide group or as methyl, ethyl or isopropyl ester during the step-by-step construction of the desired peptide structure. 



   The remaining free reactive groups that are not involved in the construction of the peptide structure can be blocked by the following special measures: The bGuanino group of the arginine is protected by NO2, Tos or by simple protonization, while the e-amino group of the lysine is protected by CbO, BOC or Tos is protected. 



   In the synthesis of the tripeptide chain, one can first add the blocking group (acyl or sulfonyl group) to the N-terminal amino acid or dipeptide acid, then activate the carboxyl group of the blocked amino acid or dipeptide acid and finally the activated amino acid derivative or dipeptide acid derivative thus obtained to complete the peptide chain attach the required dipeptide derivative. 



   The preparation of the substrate according to the invention by the methods mentioned above is described in more detail in the following examples. 



   The analysis of the eluates and products obtained according to the examples was carried out by thin layer chromatography.  Glass plates coated with silica gel F 254 (Merck) were used for this purpose.  The following solvent systems were used to develop the thin-layer chromatograms: A chloroform / methanol (9: 1) B n-propanol / ethyl acetate / water (7: 1: 2) C n-butanol / acetic acid / water (3: 1: 1)
The chromatograms were first in UV light and then by reaction with chlorine / toluidine (see G.  PATAKI, thin layer chromatography in amino acid and peptide chemistry, Walter de Gruyter & Co. , Berlin, 1966, p.     125) developed. 



  The following abbreviations are used in the present description (including patent claims): Ala = L-alanine p-AIa = B-alanine Arg = L-arginine But = L-2-aminobutyric acid 4-But = 4-aminobutyric acid Gly = glycine Ile = L -Isoleucine D-Ile = D-Isoleucine Leu = Leucine Lys = L-Lysine NLeu = L-Norleucine NVal = L-Norvaline Ser = L-Serine Val = L-Valine D-Val = D-Valine Ac = Acetyl AczO = Acetic anhydride AcOH = acetic acid BOC = tert. -Butoxycarbonyl Bz = benzoyl Bzl = benzyl BzrO = benzoic anhydride Cbo = carbobenzoxy DCCI = N, N'-dicyclohexylcarbodiimide DCHA = dicyclohexylamine DCH = N, N '-dicyclohexylurea DMF = dimethylformamide DSC = ethylamine MPTromat

     N, N, N ', N', N ", N" -hexamethylphosphoric acid triamide LMS = solvent system MCbo = p-methoxyphenylazocarbobenzoxy MeOH = methanol NA = naphthylamide OtBu = tert. -Butoxy OEt = ethoxy OisoPr = iso-propoxy OMe = methoxy OpNP = p-nitrophenoxy OSu = N-succinimidoxy pNA = p-nitroanilide Smp = melting point TFA = trifluoroacetyl Tos = p-toluenesulfonyl
example 1
I.    N "-Bz-Val-Gly-Arg-pNA. HCI
Ia) N-Cbo-Arg (NO2) -pNA
1) 32.55 g (92.1 mmol) of well-dried Cbo-Arg (NO2) -OH in 200 ml of N, N, N ', N', N ', dried and dried over P2Os were placed in a 500 ml three-necked round bottom flask. , N "-Hexamethylphosphorsäuretriamid dissolved with exclusion of moisture at 20" C. 

  First 9.32 g (92.1 mmol) of Et3N and subsequently 18.90 g (115.1 mmol) of p-nitrophenyl isocyanate in 25% excess were added to this solution in portions.  After a reaction time of 24 hours at room temperature, the reaction solution was added dropwise with stirring to 1.5 liters of 2% NaHCO3 solution.  The precipitated product was filtered off and three times with 0.7 liters of 2% NaHCO solution, three times with 0.7 liters of dest.  Water, three times with 0.5 liters of 0.5N HCI and finally three times with 0.5 liters of distilled water.  Washed water. 

  The product obtained in this way was dried in vacuo at 40 ° C. and then extracted twice with 200 ml of boiling methanol, the main amount of the N-bo-co-nitroarginine lactam obtained as a by-product being dissolved out in addition to only small amounts of the desired product.  The pre-purified crude product obtained in this way was extracted twice with 50 ml of DMF heated to 70 ° C. after drying, the desired product being completely dissolved out, while the by-product, namely N, N'-bis-p-nitrophenylurea, was undissolved stayed behind.  The DMF solution was concentrated in vacuo at 40 "C. 

  After the addition of MeOH, a substance crystallized out which was chromatographically uniform in the solvent systems A and B and had a mp.  from 186-188.5 "C. 



   Yield: 29.75 g (68.2% of theory).  The following values were determined by elementary analysis and calculation from the gross formula C20H23N707 (the values for the gross formula are given in parentheses): C = 50.42% (50.74%); H = 4.98% (4.90%); N = 20.90% (20.71%).  [o] 2u = 1.270 (c = 1.0; AcOH).    



   2) In a 500 ml three-necked round bottom flask, 17.7 g (50 mmol) of dried Cbo-Arg (NO2) -OH were dissolved in 350 ml of THF: DMF (1: 1), followed by 5.05 g (50 mmol) of Et3N with exclusion of moisture were added.  After the reaction solution had cooled to −10 ° C., 6.85 g (50 mmol) of isobutyl chloroformate, dissolved in 30 ml of THF, were then added dropwise over the course of 15 minutes, maintaining a temperature of from -10 ° to -5 ° C.  After about 10 minutes, 8.2 g (50 mmol) of p-nitroaniline, dissolved in 15 ml of DMF, were added dropwise, again maintaining a temperature of from −10 ° C. to -5 ° C.  After 2 hours the cooling was interrupted and the reaction mixture was left to stand at room temperature for 24 hours. 



  After distilling off the solvent in vacuo, the residue was successively three times with distilled water.  Water, three times with 5% NaHCO3 solution and again three times with dist.  Water digested.  After drying in vacuo, the crude product was dissolved in methanol and passed over a column of Sephadex LH-20 (crosslinked dextran gel) equilibrated with methanol.  A fraction of the eluate gave 7.67 g of substance (32.4% of theory) with the same physical properties as the end product obtained in accordance with paragraph 1). 



   3) 17.7 g (50 mmol) of Cbo-Arg (NO2) -OH was dissolved in 75 ml of DMF.  After cooling to -10 ° C., 10.3 g (50 mmol) DCCT and 8.2 g (50 mmol) p-nitroaniline were added to the solution in succession.  After 4 hours at −10 ° C. and 20 hours at 20 ° C., the DCH formed was filtered off and the filtrate was evaporated to dryness.  The crude product was dissolved in MeOH and passed through a Sephadex LH-20 column equilibrated with MeOH. 



  In addition to a large number of by-products, namely N-Cbo-Arg (NO) -N, N '- dicyclohexylurea, 4.32 g of substance (17.9% of theory) were obtained from a fraction of the eluate, which had the same physical properties as the Paragraph 1) produced end product. 



   Ib) N-Cbo-Gly-Arg (NO2) -pNA
With exclusion of moisture, 9.5 g (20 mmol) of N "-Cbo-Arg (NO1) -pNA (see Example Ia) were treated in a flask with 80 ml of 2-n HBr in glacial acetic acid with stirring for one hour at 20C, this substance detached itself under CO2 development.  The reaction solution was slowly added dropwise to 600 ml of dried ether while stirring vigorously, HBR.       H-Arg (NO2) pNA failed.  The ether phase was suctioned off with a filter rod.  The remaining precipitate was treated four times with 150 ml of dry ether in order to remove the benzyl bromide formed and excess HBr and acetic acid.  After drying in vacuo over NaOH platelets, the deblocked product was obtained in quantitative yield. 

  The dry hydrobromide derivative was dissolved in 50 ml DMF, cooled to -100C, and 4.16 ml (30 mmol) Et3N was added to release H-Arg (NO2) -pNa from the hydrobromide. 



  The Et3N HBr salt formed was filtered off with suction, washed with a little cold DMF, and 6.94 g (21 mmol) of Cbo-Gly-OpNP at -10 ° C. were added to the filtrate.  After a few hours the reaction solution had reached room temperature.  It was cooled again to −1 ° C. and buffered with 1.4 ml (10 mmol) of Et3N.  After about 5 hours, another 1.4 ml Et3N were added.  After a further 24 hours, the reaction solution was concentrated to dryness in vacuo at 40 ° C.  The residue was three times with 100 ml of dist.    H2O digested and again dried in vacuo at 40 "C over NaOH plates. 

  The dried product was recrystallized from methanol, whereby 6.77 g of substance which was chromatographically uniform in solvent systems A and B were obtained.  A further 4.29 g of substance were obtained from the mother liquor by gel filtration through a column of Sephadex LH-20 equilibrated with MeOH.  A total of 11.06 g (87.7% of theory) of uniform substance with an mp.  obtained from 159-161 "C.  Elemental analysis and calculation from the gross formula C22H26NsO8 gave the following values (the values calculated from the gross formula are in brackets): C = 50.07% (C = 49.81%); H = 4.99% (H = 4.94%); N = 21.48% (N = 21.12%). 



   Ic) Na-Cbo-Val-Gly-Arg (NO2) -pNA
10.6 g (20 mmol) Na-Cbo-Gly-Arg (NO2) -pNA (see Example Ib) were treated with 120 ml 2-n HBr in glacial acetic acid (0.24 mol) and worked up as in Example Ib).  The hydrobromide of the dipeptide derivative dried in vacuo over NaOH platelets was dissolved in 50 ml of DMF and, after cooling, 3.04 g of Et3N in 10 ml of DMF (30 mmol) were added. 



  Et3N HBr formed was filtered off and washed with a little cold DMF.  8.20 g (22 mmol) of Cbo Val-OpNP were added to the filtrate.  The further workup was carried out analogously to that in Example Ib.  By gel filtration on a Sephadex LH-20 column equilibrated with MeOH, after elution with MeOH, 10.95 g (87.0% of theory) of substance chromatographically uniform in the solvent systems A and B with an mp.  won from 212-214 "C. 



  The elementary analysis and the calculation from the gross formula C2vH3sNsOs gave the following values: C = 52.01% (51.50%); H = 5.68% (5.60%); N = 20.27% (20.02%).     (The values calculated from the gross formula are in brackets. )
Id) Na-Bz-Val-Gly-Arg (NO2) -pNA
10.90 g (17.3 mmol) of Nt-Cbo-Val-Gly-Arg (NO2) -pNA (see Example Ic) were treated with 70 ml of 2-n HBr in glacial acetic acid (0.14 mol).  The reaction mixture was worked up as described in Example Ib.  After the dried tripeptide hydrobromide derivative had been dissolved in 60 ml of DMF, 3.60 ml of Et3N (26 mmol) were added to the solution after cooling.  The Et3N HBr formed was filtered off and washed with a little cold DMF.  

  6.66 g (29.4 mmol) of benzoic anhydride were added to the filtrate obtained. 



  It was then buffered and worked up as in Example Ib).  The dried product was recrystallized from MeOH, 4.28 g of substance, which was chromatographically uniform in the solvent systems A and B, with an mp.  of 222-223 C.  From the mother liquor was still 3.75 g of substance with a mp by gel filtration through a column of Sephadex LH-20 equilibrated with MeOH.  won from 222-223 "C.  In total, this resulted in 8.03 g (77.4% of theory) of uniform substance. 



  Elemental analysis and calculation from the gross formula C26H33NsOs gave the following values: C = 51.88% (52.08%): H = 5.63% (5.55%); N = 21.56% (21.03%).    



   I) Na-Bz-Val-Gly-Arg-pNA. HCI
6.044 g (10.08 mmol) of N -Bz-Val-Gly-Arg (NO2) -pNA (see Example Id) were weighed out in a ground Erlenmeyer flask.  81 ml of 1 molar boron tris trifluoroacetate in trifluoroacetic acid were added.  After a reaction time of 2 hours at 0 C and then 22 hours at 20 "C, the nitro protective group was split off with stirring and exclusion of atmospheric moisture.  The reaction solution was concentrated to dryness in vacuo, the residue was taken up in MeOH.  In order to convert the peptide derivative into the HCl salt, 1.0 ml of conc.  HCI added and the solution evaporated to dryness in vacuo.  After repeating this operation three times, the residue was dissolved in 200 ml of 30% acetic acid. 

  For purification, the AcOH solution was applied to a Sephadex G-15 column equilibrated with 30% AcOH and eluted with 30% AcOH.  The part of the AcOH eluate which could be cleaved by trypsin treatment, with the release of p-nitroaniline, was concentrated after the addition of 0.85 ml (10.1 mmol).  HCI lyophilized.  4.75 g (79.7% of theory) of an amorphous powder were obtained, which was uniform in the DSC in LMS C.  Elemental analysis and calculation from the gross formula C26H35NsO6CI gave the following values: C = 52.71% (52.83%); H = 5.88% (5.97%); N = 19.07% (18.96%) and Cl = 5.95% (6.00%).    



   The amino acid analysis showed the expected amino acids in the correct ratio: Ar: 0.97 - Gly: 1.0 - Val: 0.98. 



   Example 2
II.    H-Val-Gly-Arg-pNA.       2HC1
630 mg (1 mmol) of N 1 -Cbo-Val-Gly-Arg (NO2) -pNA prepared according to Example 1, paragraph Ic were weighed into the reaction vessel of a Sakakibara apparatus.  10 ml of dried hydrogen fluoride gas were condensed in the reaction vessel.  After a reaction time of 1 hour at 0 C, the Na carbobenzoxy protective group and the nitro protective group of arginine were removed with stirring.  The condensed hydrogen fluoride gas was distilled off in vacuo and the residue was dissolved in DMF.  In order to convert the peptide derivative into the HCl salt, 0.2 ml of conc.    HC1 added and the solution evaporated to dryness. 

  After repeating this procedure twice, the residue was dissolved in 25 ml of 30% AcOH.  For purification, the AcOH solution was applied to a Sephadex G-15 column equilibrated with 30% AcOH and eluted with 30% AcOH.  The part of the AcOH eluate which could be cleaved by trypsin treatment, with the release of p-nitroaniline, was concentrated after the addition of 160 ul (2 mmol).    HC1 lyophilized.  360 mg (68.8% of theory) of an amorphous powder were obtained, which was uniform in the DSC in LMS C. 



  Elemental analysis and calculation from the gross formula C19H, 2NsOsC12 gave the following values: C = 43.85% (43.60%); H = 6.23% (6.16%); N = 21.65% (21.41%) and Cl = 13.42% (13.55%).    



   The amino acid analysis showed the expected amino acids in the correct ratio: Arg: 0.98 - Gly: 1.00 - Val: 0.96. 



   Example 3 III.  N -Bz-Ile-Gly-Arg-pNA HCl
IIIa) Nst-Cbo-Ile-Gly-Arg (NO2) -pNA
5.3 g (10 mmol) of the compound prepared according to Example 1, paragraph Ib, were deblocked according to Example 1, paragraph Ib, dissolved in 35 ml DMF and mixed with 2.08 ml (15 mmol) Et3N.  After cooling to −1 ° C., the Et3N HBr formed was filtered off and washed with a little cold DMF. 



  The filtrate obtained was mixed with 4.25 g (11 mmol) of Cbo-Ile OpNP and the reaction solution was further treated according to Example 1, paragraph Ib.  After gel filtration on Sephadex LH-20 in MeOH, 5.50 g (85.5% of theory) of the crystalline compound league were obtained, which melted at 197-200 C and was uniform in the LMS A and B in the DSC. 



  Elemental analysis and calculation from the gross formula C21H37N9O gave the following values: C = 51.98% (52.25%); H = 5.91% (5.79%) and N = 19.73% (19.59%).    



      IIIb) N -Bz-Ile-Gly-Arg (NO2) -pNA
1.29 g (2 mmol) of the compound prepared according to Example 3, paragraph Liga, were deblocked according to Example 1, paragraph Ib, dissolved in 15 ml DMF and mixed with 420 μl (3 mmol) Et3N.  After cooling to -10 ° C., 680 mg (3 mmol) of BzzO were added to the reaction mixture, and the reaction solution was further treated in accordance with Example 1, paragraph Ib.  After gel filtration on Sephadex LH-20 in MeOH, 1.05 g (85.6% of theory) of the amorphous compound IIIb was obtained, which was uniform in the LMS A and B in the DSC.  Elemental analysis and calculation from the gross formula C27H3> N9Os gave the following values: C = 52.54% (52.85%); H = 5.85% (5.75%) and N = 20.68% (20.54%). 



   III.    NU-Bz-Ile-Gly-Arg-pNA HCl 614 mg (1 mmol) of the compound prepared according to Example 3, Section IIIb, were weighed into the reaction vessel of a Sakakibara apparatus.  10 ml of dried hydrogen fluoride gas were condensed in the reaction vessel.  After a reaction time of one hour at 0 C, the arginine nitro protective group was split off with stirring.  The condensed hydrogen fluoride gas was distilled off from the reaction mixture in vacuo and the residue was dissolved in DMF.  In order to convert the peptide derivative into the HCl salt, 0.2 ml (-2.5 mmol) of conc.  HCI added and the solution evaporated to dryness.  After repeating this procedure twice, the residue was dissolved in 50 ml of 30% AcOH. 

  For purification, the AcOH solution was applied to a Sephadex G-15 column equilibrated with 30% AcOH and eluted with 30% AcOH.  The part of the AcOH eluate which could be cleaved by trypsin treatment, with the release of p-nitroaniline, was concentrated after the addition of 80 l (1 mmol).  HCI lyophilized.  This gave 505 mg (83.5% of theory) of an amorphous powder which was uniform in the DSC in LMS C.  Elemental analysis and calculation from the gross formula C27H37NsO6CI gave the following values: C = 53.28% (53.59%); H = 6.21% (6.16%); N = 18.70% (18.52%) and C1 = 5.82% (5.86%). 

 

   The amino acid analysis showed the expected amino acids in the correct ratio: Arg: 0.96 - Gly: 1.00 - Ile: 0.98
Example 4
IV.    N "-Bz-Leu-Gly-Arg-pNA HCl
IVa) N'l-Cbo-Leu-Gly-Arg (NO2) -pNA
2.65 g (5 mmol) of the compound prepared according to Example 1, paragraph Ib, were deblocked according to Example 1, paragraph Ib, dissolved in 25 ml DMF and mixed with 1.04 ml (7.5 mmol) Et3N.  After cooling to -1 0 C, the Et3N formed.       HBr filtered off and washed with a little cold DMF. 



  The filtrate obtained was mixed with 2.13 g (5.5 mmol) of Cbo-Leu OpNP and the reaction solution was further treated according to Example 1, paragraph Ib.  After gel filtration on Sephadex LH-20)> in MeOH, 2.85 g (88.6% of theory) of the crystalline compound IVa were obtained, which melted at 189-191 "C and was uniform in the LMS A and B in the DSC. 



  Elemental analysis and calculation from the gross formula C2sH37NsOs gave the following values: C = 52.05% (52.25 / o): H = 5.79% (5.79%) and N = 19.91% (17.59% ). 



   IVb) N -Bz-Leu-Gly-Arg (NO2) -pNA
1.29 g (2 mmol) of the compound prepared according to Example 4, paragraph IVa, were deblocked according to Example 1, paragraph Ib, dissolved in 15 ml DMF and mixed with 420 (3 mmol) Et3N.  After cooling to −1 ° C., 680 mg (3 mmol) of BzzO were added to the reaction mixture and the reaction solution was further treated in accordance with Example 1, paragraph Ib. 



  After gel filtration on Sephadex LH-20 in MeOH, 0.99 g (80.7% of theory) of the amorphous compound IVb, which was uniform in the LMS A and B in the DSC, was obtained. 



  Elemental analysis and calculation from the gross formula C27H35NOS gave the following values: C = 52.49% (52.85%); H = 5.81% (5.75%) and N = 20.59% (20.54%).    



   IV.    Nft-Bz-Leu-Gly-A} g pNA HCl
614 mg (1 mmol) of the compound prepared according to Example 4, paragraph IVb were converted to compound IV according to Example 2, paragraph II.  Purification: Gel filtration on Sephadex G-15 in 30% AcOH.    



   Yield: The part of the AcOH eluate which could be cleaved by trypsin treatment with the release of p-nitroaniline was concentrated after the addition of 80 l (1 (1 mmol). 



  HCI lyophilized.  This gave 508 mg (84.0% of theory) of an amorphous powder which was uniform in the DSC in the LMS C.  Elemental analysis and calculation from the gross formula C2-H3-NsO6CI gave the following values: C = 53.75% (53.59%): H = 6.21% (6.16%); N = 18.74% (18.52%) and Cl = 5.76o'o (5.86%).  The amino acid analysis showed the expected amino acids in the correct ratio: Arg: 0.97 - Gly: 1.00 - Leu: 1.02. 



   Example 5
V.    NQ-Bz-Val-Ser-Arg-pNA-HCl
Va) N'l-BOC-Ser (OBzl) -Arg (NO2) -pNA
3.55 g (7.5 mmol) of the compound prepared according to Example 1, paragraph Ia was deblocked according to Example 1, paragraph Ib, dissolved in 35 ml DMF and treated with 1.56 ml (11.3 mmol) Et3N.  After cooling to −100 ° C., the Et3N HBr formed was filtered off and washed with a little cold DMF.  The filtrate obtained was mixed with 3.35 g (8 mmol) of BOC-Ser (OBzl) -OpNP and the reaction solution according to Example 1, paragraph Ib, was further treated.  After gel filtration on Sephadex LH-20 in MeOH, 4.09 g (88.4% of theory) of a partially crystalline compound Va which was uniform in DSC in LMS A and B were obtained. 



  Elemental analysis and calculation from the gross formula C27H36NsO9 gave the following values: C = 52.82% (52.59%); H = 5.78% (5.88%) and N = 18.30% (18.17%). 



   Vb) NCt-BOC-Val-Ser (OBzl) -Arg (NO2-pNA
3.08 mg (5 mmol) of the compound prepared according to Example 5, paragraph Va, were weighed into an Erlenmeyer flask and treated with 15 ml of trifluoroacetic acid with exclusion of moisture.  With stirring at 20 C, 30 minutes, the substance dissolved with the evolution of CO2.     The reaction solution was slowly added dropwise to 250 ml of dried ether with vigorous stirring, CF3COOH.        H-Ser (OBzl) -Arg (NO2) -pNA failed.  The ether phase was suctioned off with a filter rod.  The remaining precipitation was treated three times with 50 ml of dry ether in order to tert the formed.  Remove butanol and excess trifluoroacetic acid. 

  After drying in vacuo over NaOH platelets, the deblocked product was obtained in almost quantitative yield. 



  The dry trifluoroacetate derivative was dissolved in 25 ml DMF, cooled to -10 ° C. and mixed with 1.04 ml (7.5 mmol) Et3N in order to release H-Ser (OBzl) -Arg (NO2) -pNA from the trifluoroacetate, and then 1.73 g (5.5 mmol) of BOC-Val-OSu was added.  After a few hours the reaction solution had reached room temperature.  It was cooled back to -10 "C and buffered with 0.35 ml (2.5 mmol) Et3N.  After about 5 hours, another 0.35 ml Et3N was added.  After a further 24 hours, the reaction solution was concentrated to dryness in vacuo at 40 ° C.  The residue was treated according to Example 1, paragraph Ib.  After gel filtration on Sephadex LH-20 in MeOH, 2.81 g (78.5% of theory) of an amorphous powder was obtained which was uniform in the LMS A and B in the DSC. 

  Elemental analysis and calculation from the gross formula C32H4sN9O10 gave the following values: C = 54.03% (53.70%); H = 6.40% (6.34%) and N = 17.88% (17.61%).    



   Vc) N -Bz-Val-Ser (OBzl) -Arg (NO2) -pNA
716 mg (1 mmol) of the compound prepared according to Example 5, paragraph Vb were deblocked according to Example 5, paragraph Vb, dissolved in 8 ml DMF and mixed with 210 p1 (1.5 mmol) Et3N.  After cooling to -10.degree. C., 450 mg (2 mmol) of BzzO were added and the reaction solution was further treated according to Example 1, paragraph Ib.  After gel filtration on Sephadex LH-20 in MeOH, 532 mg (73.9% of theory) of the crystalline compound Vc was obtained, which melted at 175-178 C and was uniform in the LMS A and B in the DCS.  Elemental analysis and calculation from the gross formula C34H41N9O9 gave the following values: C = 56.65% (56.74%); H = 5.69% (5.74%) and N = 17.83% (17.52%). 



   V.    N -Bz-Val-Ser-Arg-pNA HCl
360 mg (0.5 mmol) of the compound prepared according to Example 5, paragraph Vc were converted into compound V according to Example 2, paragraph II.  Purification: Gel filtration on Sephadex G-15 in 30% AcOH. 



   Yield: The part of the AcOH eluate which could be cleaved by trypsin treatment, with the release of p-nitroaniline, was concentrated after adding 40 l (0.5 mmol). 



  HCl lyophilized.  160 mg (51.5% of theory) of an amorphous powder were obtained, which was uniform in the DSC in LMS C.  Elemental analysis and calculation from the gross formula C27H37N807C1 gave the following values: C = 52.49% (52.21%); H = 6.08% (6.00%); N = 18.26% (18.04%) and Cl = 5.63% (5.71%). 



   The amino acid analysis showed the expected amino acids in the correct ratio: Arg: 0.99 - Ser: 0.95 - Val: 1.00. 



   Example 6
VI.    Na-Bz-Ile-Ser-Arg-pNA HCl
Vla) N -BOC-Ile-Ser (OBzl) -Arg (NO2) -pNA
1.54 g (2.5 mmol) of the compound prepared according to Example 5, paragraph Va, were deblocked according to Example 5, paragraph Vb, dissolved in 15 ml DMF and mixed with 520 kl (3.75 mmol) Et3N.  After cooling to −100 ° C., 905 mg (2.75 mmol) of BOC-Ile-OSu were added and the reaction solution was further treated in accordance with Example 1, paragraph Ib.  After gel filtration on Sephadex LH-20 in MeOH, 1.45 g (79.5% of theory) of the amorphous compound VIa was obtained, which was uniform in the LMS A and B in the DSC.  

  Elemental analysis and calculation from the gross formula C33H47N9O10 gave the following values: C = 54.82% (54.31%); H = 6.52% (6.49%) and N = 17.35% (17.27%).      



   VIb) N -Bz-Ile-Ser (OBzl) -Arg (NO2) -pNA
730 mg (1 mmol) of the compound prepared according to Example 6, paragraph VIa, were deblocked according to Example 5, paragraph Vb and dissolved in 7 ml DMF.  After cooling to -10 ° C., 210 pLI (1.5 mmol) of EtN and then immediately 450 mg (2 mmol) of BzrO were added to the solution.  The reaction solution was treated according to Example 1, paragraph Ib.  After gel filtration on Sephadex LH-20 in MeOH, 615 mg (83.8% of theory) of the amorphous compound VIb were obtained, which was uniform in the LMS A and B in the DSC.  Elemental analysis and calculation from the gross formula C35H43N9O9 gave the following values: C = 57.61% (57.29%); H = 6.01% (5.91%) and N = 17.53% (17.18%). 



   VI.    N "-Bz-Ile-Ser-Arg-pNA HCl
367 mg (0.5 mmol) of the compound prepared according to Example 6, paragraph VIb were converted to compound VI according to Example 2, paragraph II.  Purification: Gel filtration on Sephadex G-15 in 30% AcOH. 



   Yield: The part of the AcOH eluate which could be cleaved by trypsin treatment with the release of p-nitroaniline was concentrated after adding 40 l (0.5 mmol). 



  HCI, lyophilized.  168 g (45.8% of theory) of an amorphous powder were obtained, which was uniform in the DSC in the LMS C.  Elemental analysis and calculation from the gross formula C2sH3sNsO7CI gave the following values: C = 53.21% (52.95%); H = 6.26% (6.19%); N = 17.88% (17.64%) and Cl = 5.57% (5.58%). 



   The amino acid analysis showed the expected amino acids in the correct ratio: Arg: 0.96 - Ser: 0.95 - Ile: 1.00. 



   Example 7
VII.    Na-Bz-Leu-Ser-Arg-pNA-HCl
VIIa) Na-BOC-Leu-Ser (OBzl) -Arg (NO2) -pNA
1.55 g (2.5 mmol) of the compound prepared according to Example 5, paragraph Va, was deblocked according to Example 5, paragraph Vb, dissolved in 15 ml DMF and mixed with 520 (3.75 mmol) Et3N.  After cooling to −1 ° C., 0.91 g (2.78 mmol) of BOC-Leu-OSu were added and the reaction solution was further treated according to Example 1, paragraph Ib.  After gel filtration on Sephadex LH-20 in MeOH, 1.44 g (78.9% of theory) of the amorphous compound VIIa was obtained, which was uniform in the LMS A and B in the DSC.  Elemental analysis and calculation from the gross formula C33H47N9O10 gave the following values: C = 54.70% (54.31%); H = 6.55% (6.49%) and N = 17.43% (17.27%).    



   VIIb) Na-Bz-Leu-Ser (OBzl) -Arg (NO2) -pNA
1.46 g (2 mmol) of the compound prepared according to Example 7, paragraph VIIa, was deblocked according to Example 5, paragraph Vb and dissolved in 15 ml DMF.  After cooling to -0.degree. C., 420 ml (3 mmol) of Et3N and then immediately 0.90 g (4 mmol) of BzrO were added to the solution.  The reaction solution was treated according to Example 1, paragraph Ib.  After gel filtration on Sephadex LH-20 in MeOH, 1.25 g (85.2% of theory) of the amorphous compound VIIb was obtained, which was uniform in the LMS A and B in the DSC.  Elemental analysis and calculation from the gross formula C35H43N9O9 gave the following values: C = 57.38% (57.29%); H = 5.98% (5.91%) and N = 17.57% (17.18%).    



   VII.    Na-Bz-Leu-Ser-Arg-pNA-HCl
735 mg (1 mmol) of the compound prepared according to Example 7, paragraph VIIIb were converted into compound VII according to Example 2, paragraph II.  Purification: Gel filtration on Sephadex G-15 in 30% AcOH. 



   Yield: The part of the AcOH eluate which could be cleaved by trypsin treatment, with the release of p-nitroaniline, was concentrated after addition of 80 (1 mmol). 



  HCI lyophilized.  355 mg (55.9% of theory) of an amorphous powder were obtained, which was uniform in the DSC in the LMS C.  Elemental analysis and calculation from the gross formula C2sH39NsO7Cl gave the following values: C = 53.09% (52.95%); H = 6.09% (6.19%); N = 17.91% (17.64%) and Cl = 5.49% (5.58%). 



   The amino acid analysis showed the expected amino acids in the correct ratio: Arg: 0.95 - Ser: 0.93 - Leu: 1.00. 



   Example 8
VIII.    Na-3-phenylpropionyl-Val-Gly-Arg-pNA-HCl
VIIIa) Na-3-phenylpropionyl-Val-Gly-Arg (NO2) -pNA
3.15 g (5 mmol) of the compound prepared according to Example 1, paragraph Ic, was deblocked according to Example 1, paragraph 1b and dissolved in 25 ml of DMF.  After cooling to -10 ° C., 1.05 ml (7.5 mmol) of Et3N and then 1.50 g (5.5 mmol) of 3-phenylpropionic acid p-nitrophenyl ester were added to the solution.  The reaction solution was treated according to Example 1, paragraph Ib.  After gel filtration on Sephadex LH-20 in MeOH, 2.75 g (87.6% of theory) of crystalline compound VIIIa was obtained, which melted at 216-218 "C and was uniform in the LMS A and B in the DSC. 

  Elemental analysis and calculation from the gross formula C2sH37N9Os gave the following values: C = 53.38% (53.58%); H = 6.02% (5.94%) and N = 20.40% (20.09%). 



   VIII.    N'-3-phenylpropionyl-Val-Gly-Arg-pNA.     HCI
1.26 g (2 mmol) of the compound prepared according to Example 8, paragraph VIIIa were converted into compound VIII according to Example 1, paragraph 1.  Cleaning: Gel filtration on Sephadex G-15 in 30% AcOH.    



   Yield: The part of the AcOH eluate which could be cleaved by trypsin treatment, with the release of p-nitroaniline, was concentrated after the addition of 160 u1 (2 mmol). 



  HCI lyophilized.  1.12 g (90.4% of theory) of an amorphous powder were obtained, which was uniform in the DSC in the LMS C.  Elemental analysis and calculation from the gross formula C2sH39NsO6Cl gave the following values: C = 54.58% (54.32%); H = 6.29% (6.35%); N = 18.40% (18.10%) and Cl = 5.67% (5.73%).    



   The amino acid analysis showed the expected amino acids in the correct ratio: Arg: 0.96 - Gly: 1.00 - Val: 1.02. 



   Example 9 IX.       Na-2-phenylacetyl-Val-Gly-Arg-pNA-HCl
IXa) Na-2-phenylacetyl-Val-Gly-Arg (NO2) -pNA
1.57 g (2.5 mmol) of the compound prepared according to Example 1, paragraph Ic, were deblocked according to Example 1, paragraph Ib and dissolved in 15 ml of DMF.  After cooling to −10 ° C., 520 μl (3.75 mmol) of Et3N and then 710 mg (2.75 mmol) of 2-phenylacetic acid pnitrophenyl ester were added to the solution.  The reaction solution was treated according to Example 1, paragraph Ib.  After gel filtration on Sephadex LH-20 in MeOH, 1.35 g (88.0% of theory) of the crystalline compound IXa was obtained, which melted at 180-184 "C and was uniform in the LMS A and B in the DSC.  

  Elemental analysis and calculation from the gross formula C21H35N9O5 gave the following values: C = 53.09% (52.85%); H = 5.82% (5.75%) and N = 20.91% (20.54%).   



   IX.    N? -2-phenylacetyl-Val-Gly-Arg-pNA. HCI
615 mg (1 mmol) of the compound prepared according to Example 9, paragraph IXa, were converted into compound IX according to Example 1, paragraph 1.  Purification: Gel filtration on Sephadex G-15 in 30% AcOH. 



   Yield: The part of the AcOH eluate which could be cleaved by trypsin treatment, with the release of p-nitroaniline, was concentrated after the addition of 80 l (1 mmol).    



  HCI lyophilized.  515 mg (85.1% of theory) of an amorphous powder were obtained, which was uniform in the DSC in the LMS C.  Elemental analysis and calculation from the gross formula C27H37NsO6CI gave the following values: C = 53.19% (53.59%); H = 6.21% (6.16%); N = 18.77% (18.52%) and Cl = 5.75% (5.86%). 



   The amino acid analysis showed the expected amino acids in the correct ratio: Arg: 0.94 - Gly: 1.00 - Val: 0.98. 



   Example 10
X.    Nt-Cbo-Val-Gly-Arg-pNA. HCI
Xa) N <'-Cbo-Arg-pNA HCl
In a three-necked round bottom flask of 250 ml content
16.0 g (47.0 mmol) of Cbo-Arg-OH dried in vacuo over P2O5. HCl dissolved in 90 ml absolute HMPTA with exclusion of moisture at 20 "C.  At room temperature, a solution of 4.74 g (47.0 mmol) of Et3N in 10 ml of HMPTA and then 16.4 g (100 mmol) of p-nitrophenyl isocyanate (100% excess) were added in portions to the solution obtained.  After a reaction time of 24 hours at 20 C, the HMPTA was largely distilled off in vacuo.  The residue was extracted several times with 30% AcOH.  The residue was discarded. 

  The combined acetic acid extracts were applied to a Sephadex G-15 column equilibrated with 30% AcOH for further purification and eluted with 30% AcOH.  The fraction of the AcOH eluate which could be cleaved by trypsin treatment with the release of p-nitroaniline was freeze-dried.  12.6 g of an amorphous powder were obtained, which was uniform in the DSC in the LMS C.  Elemental analysis and calculation from the gross formula C2OH2sN6OsCl gave the following values: C = 51.29% (51.67%); H = 5.48% (5.42%); N = 17.92% (18.08%); Cl = 7.50% (7.63%).    



   Xb.    Na-Cbo-Gly-Arg-pNA H Br
With exclusion of moisture, 4.65 g (10 mmol) of the compound Xa were treated with 40 ml of 2N HBr in glacial acetic acid with stirring at 20 ° C. for 45 minutes.  The amino acid derivative dissolved under CO2 development.     The reaction solution was added dropwise to 250 ml of absolute ether with vigorous stirring, where (2HBr). H-Arg pNA failed.  The ether phase was suctioned off, after which the solid phase was washed four times with 100 ml of absolute ether in order to remove the benzyl bromide formed as a by-product and the excess HBr and AcOH.  The deblocked product was obtained in quantitative yield by drying in vacuo over NaOH platelets.  The dry (2H Br). H-Arg-pNA was dissolved in 25 ml DM F. 



  1.40 ml (10 mmol) of Et3N were added to the solution, which was cooled to −100 ° C.  A precipitate of Et3N HBr was formed, which was filtered off and washed with a little cold DMF.  3.65 g (11 mmol) of Cbo-Gly-OpNP at −10 C were added to the filtrate.  After a few hours the temperature of the reaction solution had risen to 20 ° C. 



  The solution was cooled again to −10 ° C. and buffered with 0.35 ml (2.5 mmol) of Et3N.  After a further 16 hours, a further 0.35 ml of Et3N were added at −10 ° C.  After a further 24 hours, the reaction solution was concentrated to dryness in vacuo at 40.degree.  The residue was dissolved in 33% AcOH.  The solution was purified by gel filtration on a Sephadex G-15 column equilibrated with 33% AcOH.  The fraction of the AcOH eluate which could be cleaved by trypsin treatment with the release of p-nitroaniline was concentrated to dryness in vacuo. 



   The residue was dissolved in 30 ml of MeOH and added dropwise to 300 ml of absolute ether with vigorous stirring, Na-Cbo-Gly-Arg-pNA. HBr failed.  The ether phase was suctioned off, after which the solid phase was washed twice with 100 ml of absolute ether in order to remove MeOH and traces of AcOH.  After drying in vacuo over NaOH platelets, 4.45 g (78.6% of theory) of an amorphous powder were obtained which was uniform in the DSC in the LMS C.  Elemental analysis and calculation from the gross formula C22H2sN706Br gave the following values: C = 46.33% (46.65%) H = 5.04% (4.98%); N = 17.88% (17.31%) and Br = 14.20% (14.11%).    



   Xc.    Na-Cbo-Val-Gly-Arg-pNA. HCI
2.85 g (5 mmol) of compound Xb were deblocked according to paragraph Xb and dissolved in 20 ml of DMF.  After cooling to -I 0 C, 0.70 ml (5 mmol) of Et3N and then 2.05 g (5.5 mmol) of Cbo-Val-OpNP were added to the solution. 



  The reaction solution was treated according to paragraph Xb. 



   Purification: Gel filtration on Sephadex G-15 in 33% AcOH.  Yield: The part of the AcOH eluate which could be cleaved by trypsin treatment with the release of p-nitroaniline was concentrated after adding 400 l (5 mmol). 



  HCI freeze-dried.  2.55 g (82.1% of theory) of an amorphous powder were obtained, which was uniform in the DSC in the LMS C.  Elemental analysis and calculation from the gross formula C27H37NsO7CI gave the following values: C = 52.12% (52.21%); H = 6.16% (6.00%); N = 18.48% (18.04%) and Cl = 5.63% (5.71%).    



   The amino acid analysis showed the expected amino acids in the correct ratio: Arg: 1.01 - Gly: 1.00 - Val: 0.97. 



   Example 11
XI.    H-D-Val-Gly-Arg-pNA-2HCl
XIa) N -Cbo-D-Val-Gly-Arg-pNA-HCl
2.85 g (5 mmol) of the compound prepared according to Example 10, paragraph Xb, were deblocked according to Example 10, paragraph Xb and dissolved in 20 ml DMF.  After cooling to -10 C, 0.70 ml (5 mmol) of Et3N and then 2.05 g (5.5 mmol) of Cbo-D-Val-OpNP were added to the solution.  The reaction solution was treated according to Example 10, paragraph Xb. 



   Purification: Gel filtration on Sephadex G-15 in 33% AcOH.  Yield: The part of the AcOH eluate which could be cleaved by trypsin treatment with the release of p-nitroaniline was concentrated after adding 400 l (5 mmol). 

 

  HCI freeze-dried.  2.45 g (78.9% of theory) of an amorphous powder were obtained, which was uniform in the DSC in the LMS C.  Elemental analysis and calculation from the gross formula C27H37NsO7CI gave the following values: C = 51.95% (52.21%); H = 6.10% (6.00%); N = 18.33% (18.04%) and Cl = 5.60% (5.71%).    



   XIc) H-D-Val-Gly-Arg-pNA 2HCl
620 mg (1 mmol) of N -Cbo-D-Val-Gly-Arg-pNA-HCl prepared according to paragraph XIa were deblocked according to example 10, paragraph Xb. 



   Purification: Gel filtration on Sephadex G-l 5 in 33% AcOH.  Yield: The part of the AcOH eluate which could be cleaved by trypsin treatment with the release of p-nitroaniline was concentrated after the addition of 160 u1 (2 mmol). 



   HCI freeze-dried.  360 mg (68.8% of theory) of an amorphous powder were obtained, which was uniform in the DSC in the LMS C.  Elemental analysis and calculation from the gross formula C lsH32HsOsCl2 gave the following values: C = 42.98% (43.60%); H = 6.24% (6.16%); N = 21.88% (21.41%) and Cl = 13.12% (13.55%).    



   The amino acid analysis showed the expected amino acids in the correct ratio: Arg: 0.96 - Gly: 1.00 - D-Val: 0.98. 



   Example 12
XII.  Bz-Val-Gly-Arg-DPA
XIIa) Bz-Val-Gly-Arg-OH. HCl
5.9 g of compound I (Example 1) were dissolved in 200 ml of pH 8.2 buffer.  2000 NF units of trypsin were added to the solution in order to split off the p-anilino group without racemization, the pH being kept constant by dropwise addition of 1N sodium hydroxide solution.  After the reaction, 40 ml of conc.  Acetic acid added to inactivate the trypsin. 



  The solution was evaporated to dryness.  The residue was dissolved in 50 ml of 33% acetic acid and the solution was chromatographed on a Sephadex G-15 column equilibrated with 33% acetic acid.  4.43 g (94.1%) of an amorphous substance which was uniform in the DSC in the LMS C were obtained from the first fraction. 



   The following values were determined by elemental analysis and calculation from the gross formula C20H3lN6OsCl (the values for the gross formula are given in parentheses): C = 50.48% (51.01%); H = 6.71% (6.63%); N = 18.17% (17.85%); Cl = 7.38% (7.53%). 



      XVllb)
In a three-necked round-bottomed flask of 100 ml content, 942 mg (2 mmol) of the dried compound XIIa in a mixture of 7.5 ml of freshly distilled anhydrous
DMF and 15 ml of absolute THF dissolved at 20C.  280 μl (2 mmol) of Et3N were added to the solution, which had been cooled to −100 ° C., with stirring and exclusion of moisture.  A solution of 273 mg (2 mmol) of isobutyl chloroformate in was then added to the mixture within 20 minutes
5 ml of THF were added dropwise, the reaction temperature never being allowed to rise above -5 "C. 

  After an additional reaction time of 10 minutes at a temperature of - 10 "C to -5 C, the reaction mixture was a
Solution of 418.4 mg (2 mmol) of dimethyl 5-aminoisophthalate in 5 ml of DMF was added dropwise over the course of 30 minutes, the reaction temperature always being kept below -5 ° C.  The reaction mixture was left to react further at -5 ° C. for 1 hour.  It was stirred overnight at 20 "C and then cooled to - I 5 C to give the Et3N.       To let HCI crystallize.  The Et3N HCl formed was filtered off and washed with a little cold DMF. 

  The filtrate together with the washing solution was evaporated to dryness in vacuo at 50 ° C.  The
The residue was dissolved in 10 ml of 33% AcOH and by
Gel filtration on an equilibrated with 33% AcOH
Sephadex G-15 column cleaned.  The main fraction of the AcOH eluate, which could be cleaved by trypsin treatment with the release of dimethyl 5-aminoisophthalate, was concentrated to dryness in vacuo at 40 ° C.  After drying the residue in a vacuum drying cabinet at 50 ° C. over P2Os, 636 mg (48.0% of theory) of the amorphous compound XII were obtained, which in the DSC in
LMS C was uniform. 

  Elemental analysis and calculation from the gross formula C3oH40N? OsCl gave the following values: C = 54.09% (54.42%); H = 6.16% (6.09%), N = 15.07% (14.81%) and Cl = 5.15% (5.35%).    



   A number of additional substrates were synthesized according to the methods described in the examples. 



  The results of these syntheses are summarized in the table below. 



  Example end product starting materials (mmol) method elemental analysis amino acid analysis
Yield found  % ber.  % O / o 13 Bz-Val-Gly-Arg-2NA. HCI Association  XIIa (2mmol) B. 12 C 59.88 60.44 Val: Gly: Arg - H 6.47 6.43 1.02: 1.00: 0.97 ss-naphthyiamine (2 mmol) 42.4 N 16.45 16.45 Cl 5.83 5.95 14 Bz-Val-Gly-Arg-4-MeO-XIIa (2 mmol) B. 12 C 59.06 59.46 Val:

  Gly-Arg
2NA. HCL-H 6.50 6.44 1.01: 1.00: 0.98
4-methoxy-2-naphthyl-amine 38.7 N 15.83 15.66 (2 mmol) Cl 5.55 5.66 15 Cbo-Ala-Gly-Arg-pNA.  2HBr. H-Gly-Arg-pNA B. 10c C 46.76 47.10 Ala: Gly: Arg
HBr (2 mmol) - H 5.28 5.22 1.01: 1.00: 0.99
Cbo-Ala-OpNP (2.2 mmol) 82 N 17.75 17.58
Br 12.38 12.43 16 Cbo-Nleu-Gly-Arg-pNA.  2HBr. H-Gly-Arg-pNA B. l0c C 49.19 49.40 Nleu: Gly: Arg
HBr (2mmol) - H 5.82 5.78 1.02: 1.00: 1.00 Cbo-Nleu-OpNP (2.2mmol) 79.4N 16.68 16.49
Br 11.58 11.76 17 Cbo-But-Gly-Arg-pNA.  2HBr. H-Gly-Arg-pNA B. 10c C 47.73 47.93 But: Gly: Arg
HBr (2 mmol) - H 5.45 5.41 1.01: 1.00: 0.98
Cbo-But. OpNP (2.2 mmol) 80.6 N 17.40 17.20
Br 12.11 12.26 18 Ac-Val-Gly-Arg-pNA. HCI Association II (2 mmol) B. 12 C 47.53 47.68 Val: Gly:

  : Arg
Acetic anhydride - H 6.33 6.29 1.01: 1.00: 0.98 (2.2 mmol) 82.4 N21.38 21.18
Cl 6.58 6.70 Example end product starting material (mmol) method elementary analysis amino acid analysis
Yield found  % ber.  %%
19 Octanoyl-Val-Gly-Arg Comp. II (2 mmol) B. 12 C 52.45 52.89 Val: Gly: Arg pNA. HCl Octanoylchloride - H 7.47 7.40 1.00: 1.00: 0.98 (2.2 mmol) 82.5 N 18.61 18.28
Cl 5.57 5.78 20 Stearoyl-Val-Gly-Arg Comp. II (2 mmol) B. 12 C 59.43 58.98 Val: Gly: Arg pNA. HCl stearoyl chloride - H 8.79 8.70 1.01: 1.00: 0.98 (2.2 mmol) 65.4 N 15.02 14.87
Cl 4.62 4.71 21 Cbo-Gly-Val-Gly-Arg-pNA.    Association II (2 mmol) B. 12 C 51.07 51.36 Val: Gly: Arg
HCI Cbo-Gly-OpNP (2.2 mmol) - H 6.03 5.95 1.02: 1.00: 1.00
83.7 N 18.83 18.59
Cl 5.18 5.23 22 H-Gly-Val-Gly-Arg-pNA. 

  Association XXI (2 mmol) B.  11 C 43.28 43.45 Val: Gly: Arg
2HCl HBr / AcOH2N (10mmol) - H 6.11 6.08 1.01: 2.00: 0.99
91. 4 N21.98 21.72 C112.05 12.22 23 Cbo-NH- (CH2) 7-CO-Val-Arg-Verbd. II (2mmol) B. 12 C 54.76 55.14 Val: Gly: Arg pNA. HCI Cbo-NH (CH2) 7-CO-OpNP-H 6.93 6.88 1.02: 1.00: 1.01 (2.2 mmol) 79.4 N 16.75 16.54
Cl 4.60 4.65 24 NH2 (CH2) 7-CO-Val-Gly Comp. XXIII (2mmol) B. l 1 C 48.58 48.79 Val: Gly: Arg Arg-pNA. 2HC1 2N HBr / AcOH - H 7.20 7.13 1.01: 1.00: 0.98 (10 mmol) 85.6 N 19.21 18.97 C110.51 10.67 25 cyclohexylcarbonyl compound. II (2 mmol) B. 12 C 51.95 52.30 Val: Gly: Arg
Val-Gly-Arg-pNA. HCl Cyclohexylcarbon- - H 7.01 6.92 1.02: 1.00:

  : 0.99 acid-OpNP 81.2 N 19.04 18.77 (2.2 mmol) Cl 5.86 5.94 26 Cbo-NH-CH2-H -CO- Verbd. II (2 mmol) B. 12 C 54.88 55.29 Val: Gly: Arg Val-Gly-Arg-pNA. HCl Cbo-NH-CH2- - H 6.68 6.63 1.01: 1.00: 1.01
OpNP (2.2 mmol) 75.6 N 16.81 16.58
Cl 4.60 4.66 27 NH2-CH2 - # - CO-Val- Verbd. XXVI (2 mmol) B. l 1 C 48.74 48.94 Val: Gly: Arg Gly-Arg-pNA. 2HC1 2N HBr / AcOH - H 6.91 6.85 0.99: 1.00: 0.98 (lOmMol) 88.4 N 19.33 19.03 C110.58 10.70 28 Cbo-NHeCO-Val- Association III (2 mmol) B. 12 C 54.28 54.72 Val: Gly: Arg
Gly-Arg pNA. HCl Cbo-NH-CO-OpNP - H 6.53 6.48 1.02: 1.00:

  : 0.98 (2.2 mmol) 74.9 N 17.02 2. 16.89
Cl 4.67 4.75 29 H2N - # - CO-Val-Gly-Arg-Verbd. XXVIII (2 mmol) B.  11 C 48.06 48.15 Val: Gly: Arg pNA. 2HCl 2N HBr / AcOH - H 6.72 6.68 1.00: 1.00: 0.98 (10 mmol) 85.7 N 19.58 19.44
Cl10.68 10.93 30 CH3 - # - CO-Val-Gly-Arg-Verbd. II (2 mmol) B. 12 C 53.28 53.59 Val: Gly: Arg pNA. H 4-Methylbenzoyl- - H 6.21 6.16 0.99: 1.00: 0.97 chloride (2.2 mmol) 74.5 N 18.68 18.52
Cl 5.89 5.86 31 Cbo-NH-QO-CO-Val-Gly-Verbd. II (2mmol) B. 12 C 54.85 55.17 Val: Gly: Arg
Arg-pNA. HCl Cbo-NH- -CO-OpNP - H 5.76 5.72 0.98: 1.00: 0.98 (2.2 mmol) 67.8 N 17.33 17.03
Cl 4.71 4.79 32 HN-O-CO-Val-Gly Comp. XXXI (2 mmol) B. l 1 C 48.40 48.60 Val: Gly:

  : Arg Arg-pÇA. 2HC1 2N HBr / AcOH - H 5.82 5.80 0.99: 1.00: 0.97 (2.2 mmol) 83.0 N 19.80 19.12 C110.81 11.04 Example end product starting materials (mmol ) Elementary analysis method amino acid analysis
Yield found  % ago.  %% 33 Cbo-NH-CH2 j) -CO- conn. II (2 mmol) B. 12 C55.5l 55.73 Val: Gly: Arg Val-Gly-Arg-pNA-HC1 Cbo-NH-Ch2- -CO- - H 5.93 5.88 1.00: 1.00: 0.98 OpNP (2.2 mmol) 76.3 N 16.99 16.71
Cl 4.58 4.70 34 N2N-CH2 - CO-Val Comp. 

  XXXIII (2 mmol) B. l 1 C 49.12 49.39 Val: Gly: Arg
Gly-Arg pNA. 2HC1 2N HBr / AcOH - H 6.05 5.99 0.99: 1.00: 0.99 (lOmMol) 70.9 N 19.43 19.20 C110.50 10.80 35 4-phenylbutyroyl compound II (2 mmol) B. 12 C 54.88 55.01 Val: Gly: Arg Val-Gly-Arg-pNA. HC1 4-phenylbutyryl- - H 6.55 6.53 1.01: 1.00: 0.98
OpNP (2.2 mmol) 77.3 N 17.81 17.70
Cl 5.48 5.60 36 Tos-Val-Gly-Arg-pNA.    Association II (2mmol) B. 12 C 48.60 48.71 Val: Gly: Arg
HCl Tosylchloride - H 5.83 5.82 1.02: 1.00: 0.99 (2.2 mmol) 80.5 N 17.88 17.48
Cl 5.45 5.53 37 benzenesulfonyl-val. II (2 mmol) B. 12 C 47.63 47.88 Val: Gly: Arg Gly-Arg-pNA. HCl benzenesulfonyl- - H 5.66 5.63 1.00:

   1.00: 0.97 chloride (2.2 mmol) 84.1 N 17.89 17.87
Cl 5.53 5.65 38 ss-Naphtalinsulfonyl-Verbd. II (2 mmol) B. 12 C 51.14 51.44 Val: Gly: Arg Val-Gly-Arg-pNA. HC1 2-naphthalenesul- - H 5.55 5.51 0.99: 1.00: 0.99 chloride (2.2 mmol) 78.3 N 16.78 16.55
Cl 5.18 5.24 39 Methanesulfonyl-Val Comp. II (2 mmol) B. 12 C 42.29 42.51 Val: Gly: Arg
Gly-Arg-pNA methanesulfonyl- - H 5.94 5.89 0.98: 1.00: 1.01 chloride (2.2 mmol) 75.5 N 20.01 19.83
Cl 6.11 6.27 40 Aethanesulfonyl-Val Comp. II (2 mmol) B. 12 C 43.28 43.56 Val: Gly: Arg Gly-Arg-pNA. HCl Aethanesulfonyl- - H 6.13 6.09 0.98: 1.00:

  : 0.98 chloride (2.2 mmol) 71.4 N 19.55 19.35
Cl 6.03 6.12
The substrates according to the invention, e.g. B.  the substrate obtained according to Example 1, namely N-Bz-Val-Gly-Arg-pNA HCl, was used to determine various enzymes in blood plasma.  The determination was based on the principle that the cleavage product (NH2-R2) formed by the enzymatic hydrolysis of the substrate has a UV spectrum which differs from that of the substrate and is shifted to higher wavelengths.  Thus, the substrate according to Example 1, i. H.  N -Bz-Val-Gly-Arg p NA.     HCI, an absorption maximum at 302 nm (nonameter) and a molar extinction coefficient of
12950 on. 

  The absorption of the substrate is practically zero at 405 nm.  p-Nitroaniline, the cleavage product (NH2R2) formed during the enzymatic hydrolysis of the substrate, has an absorption maximum at 380 nm and a molar
Extinction coefficient of 13200.  At 405 nm the decreases
Extinction coefficient little, i.e. H.  on 9650. 



   The degree of enzymatic hydrolysis of the substrate, which is proportional to the amount of p-nitroaniline cleaved off, can easily be determined by spectrophotometric measurement at 405 nm.  The excess
The substrate does not interfere with the measurement at 405 nm.  The ratios are practically identical for the other substrates according to the invention which contain a p-nitroanilino group as a chromogenic group.  The spectrophotometric measurement was therefore carried out at 405 nm in all of these cases. 



   The enzymatic hydrolysis reaction can be shown schematically as follows:
EMI11. 1
 E = enzyme S = substrate ES = enzyme-substrate complex Pl and P = products kl, k2, k3 and k4 = rate constants dissociation constant for ES = L2 = Km (Michaelis constant if [S] [E] and k4 k3, the following applies:
EMI11. 2nd
   The rate constant at which Pt is formed is:

   v = k3. [ES] k3 k [S] (2) If E is completely bound to S, then: [ES] = [E] and v = vmax = k3 [E] (3) Lineweaver-Burk equation: Km 1 + 1 (4) vmax [S] vmax
From equation (2) it follows that the constants Km and k> determine the effectiveness of the substrate for a given enzyme.  The following method is used to determine these constants:
The enzyme and the substrate are mixed in a buffer solution and the hydrolysis reaction is followed for 2 to 30 minutes.  The concentration of the substrate [S] is varied while the enzyme concentration is kept constant. 



  If the extinction (OD = optical density) is plotted as a function of time in a coordinate system, a curve is obtained whose tangent at the zero point corresponds to the ideal course of the hydrolysis.  With the help of this tangent the initial difficulty of the hydrolysis can be determined. 



  If 1 is plotted as a function of v [S], a Lineweaver-Burk diagram is obtained (see short textbook on the biochemistry of P.  KARLSON, Georg Thieme-Verlag, Stuttgart, 1967, page 70), from which vmax and km can be determined graphically. 



     
Vmax Km and k3 = #E
E were using the substrates according to the invention, for. B.    N "-Bz-Val-Gly-Arg-pNA HCl (substrate I according to Example 1) for urokinase and trypsin.  The
Results are summarized in Table I. 



   Table 1
Urokinase activity, measured using a substrate according to the invention substrate KmMol / liter vm; lxLIMol / min.    



     1 6.06 x 10- 7.27 x 10-5CTA trypsin activity, measured using a substrate Xl according to the invention 1.83 x 10-5 4.17x 10- NF
The by means of the other substrates according to the invention for
The urokinase and trypsin values of Km and vmax are of the same order of magnitude, which shows that the substrates mentioned have a particularly high susceptibility to urokinase and trypsin. 



   In order to determine the kinetic constants Km and vmax given in Table I, aqueous dilution series of the substrates with concentrations of 0.1 to 2 FMol / ml were prepared.  2 ml of Tris imidazole buffer with pH 8.4 and ionic strength 0.30 at 37 ° C. were introduced into a measuring cuvette, after which 0.25 ml of an aqueous urokinase solution with a concentration of 400 CTA / ml was first added.  The mixture was pre-incubated at 37 "C for 1 minute.  0.25 ml of substrate solution was then added to the pre-incubated mixture.  The course of the enzymatic hydrolysis of the substrate was monitored for the various substrate concentrations using a photometer at 405 nm for 10 minutes. 

  The value of the amount of p-nitroaniline formed per minute is a measure of the rate of hydrolysis for a given substrate concentration.  The reciprocal values of the substra concentrations were plotted on the abscissa in a coordinate system, and the associated reciprocal values of the hydrolysis rate were plotted on the ordinate.  In this so-called 



  A line weaver-Burk diagram gave straight curves for the substrates listed in Table I, which was evidence that the hydrolysis of the substrates by urokinase followed Michaelis-Menten's law and the substrates are therefore ideal for the determination of urokinase.  The kinetic constants Km and vmax listed in Table I were determined from the intersection of the curves with the abscissa and the ordinate. 



   Table II 1 µM pNA per minute from substrate 1 Corresponding other units urokinase 20250 CTA urine kallikrein 1688 BAEE human thrombin 2174 NIH human plasmin 6850 CE bovine trypsin 36.6 NF
BAEE (Benzoyl-L-arginine ethyl ester) - I BAEE unit
Urine kallikrein is the amount of enzyme that hydrolyzes 1 µmol of ethyl benzoyl-L-arginine per minute under standard conditions. 



   NIH is a unit standardized by the US National Institute of Health. 



   CE = casein unit measured on casein under standard conditions. 



   NF = trypsin unit = the amount of enzyme which causes an absorption change A OD = 0.003 per minute, measured on ethyl benzoyl-L-arginine, under standard conditions (see The National Formulary XII, published by The American Pharmaceutical Association, Washington, D. C. , 1965, pages 417-418). 

 

   In the first column of Table II the amount of various enzymes is given that the inventive
Substrate I at 37 "C, pH 8.4 and ionic strength 0.3 and at one
Substrate concentration of 104 M cleaves at a rate of I µM per minute.  The corresponding amount of enzyme is given in other enzyme units in the second column.  It can be seen from Table II that the urine kallikrein enzyme present in the urine together with urokinase cleaves substrate I only slightly and therefore does not interfere with the determination of urokinase in the urine.  Thrombin also cleaves substrate I very little and unspecifically.   



   Table III
Urokinase activity, measured by means of the substrates according to the invention at a constant substrate and urokinase concentration, at 37 ° C., pH 8.4 and ionic strength 0.3 substrate
Nanomole 1 pNA II 0.92 pNA III 3.21 pNA IV 1.00 pNA V 2.82 pNA Vl 2.91 pNA VII 1.73 pNA VIII 1.32 pNA IX 1.13 pNA X l, l2pNA XI 0.20 pNA XII 2.88 XIII 2.41 XIV 2.19 XV 0.88 XVI 0.41 XVII 0.29 XVIII 1.75 XIX 1.66 XX 1.40 XXI 3.21 XXII 2.93 XXIII 2.77 XXIV 2.48 XXV 3.58 XXVI 1.99 XXVII 1.64 XXVIII 1.75 XXIX 2.22 XXX 3.39 XXXI 2.55 XXXII 2.11 XXXIII 3.08 XXXIV 2.67 XXXV 2.01 XXXVI 2, 59 XXXVII 2.71 XXXVIII 2.48 XXXIX 2.89 XL 3.01
In the attached drawing it is

   FIG. 1 shows a graphic representation in which the change in the optical density A OD, which is caused by the hydrolytic effect of different amounts of urokinase on the substrate I within 10 minutes, is plotted as a function of the amount of urokinase in a coordinate system.  It can be seen from this figure that urikinase concentrations in the range from 1 to 10 CTA / ml can be determined exactly.  When determining the urokinase content in the urine of 14 healthy test persons, values of about 8 CTA units per ml urine were found. 



   The determination of urokinase in urine is particularly important because, as mentioned above, pathological conditions can be determined relatively easily and quickly.  The most widely used method for
The determination of urokinase in urine is the so-called  Fibrin plate method [I.  Bjerrehuus, Scand.  J.  Clin.  Lab.  Invest.  4,179 (1952); F.  E.  Smyrniotis et al.     Thromb.  Diath.  et Haemorrh. 



  Vol.  III, 258 (1959)], which is carried out as follows: According to the method of Mullertz jActa physiol.  Scan.  26, 174 (1954)], fibrin plates (fibrinogen content: 0.2%) are produced. 



  For each urokinase determination, a fibrin plate and 30 lambda drops (lambda drop pipette) of undiluted, 1: 2 and 1: 4 diluted urine are used (if the urokinase concentration is very high, the dilution factors can be doubled).  The plates are incubated at 30 "C for 16 hours.  The lysed zones are made visible by adding a drop of Congo red (0.1%).  The product of two diameters perpendicular to each other (the lysed zones) serves as a measure of the lysed zones.  The urokinase concentration (units per ml) is determined using a comparison curve, which is established using a dilution series with standard urokinase. 



   In this method, the urokinase concentration is not determined directly, but via the amount of plasmin formed from plasminogen by urokinase.  This determination of end products via a chain of reactions is fraught with many sources of error.  Other serious disadvantages of this method are that the incubation time is very long and that a calibration curve has to be drawn up for each determination.  The evaluation of the results is also relatively complicated and inaccurate due to the non-linearity of the calibration curve. 



   By means of the substrates according to the invention, the disadvantages of the determination method described above can be overcome smoothly, since the urokinase concentration is measured directly and no side reactions falsify the measurement result.  In addition, the measurement results are available after only 10 minutes, which is particularly important for clinical diagnostics.  Another advantage is that the measurement results can be given in substrate units.  A change in the optical density A OD of 0.010 / min.     corresponds to a content of 1 milliunit urokinase per ml content of the test batch. 



   When it comes to determining plasminogen activators in blood plasma, it is often expedient to carry out the determination in a buffer solution which contains aprotinin and / or hirudin, since the latter do not inhibit the plasminogen activators, but others do. any activated plasma enzymes, such as. B.  Inhibit thrombin, plasmin and plasma kallikrein, which could interfere with the determination of the plasminogen activators under certain circumstances.  You can e.g. B.  a tris-imidazole or glycine
Use buffers with a pH of 8.2 to 8.6 and an ionic strength of 0.15 to 1.0, to which 0.02 to 0.2 trypsin inhibitor units aprotinin and / or 0.001 to
10 antithrombin units hirudin added per ml. 



   If the urokinase determination has to be carried out with body fluids or tissue extracts containing inhibitors of urokinase, it may be expedient to remove the body fluids or  Add an excess of urokinase to tissue extracts, incubate the mixture for a few minutes and determine the remaining urokinase activity by adding the substrate and measuring the amount of the cleavage product. 



   It was also surprisingly found that the substrates according to the invention are also very sensitive to trypsin because of their special conformation and are catalytically cleaved by the latter at unexpectedly high speeds.  The new substrates are particularly well suited for the determination of trypsin in duodenal juice and in the blood in acute pancreatitis, in which trypsin enters the bloodstream, but only occurs in low concentrations due to the high blood volume.  The known substrates hitherto used for the determination of trypsin are far too insensitive for the determination of such low concentrations. 



   The determination of trypsin in duodenal juice can be carried out, for example, as follows: 1.79 ml of tris-imidazole buffer with a pH of 8.4 and an ionic strength of 0.30 at 37 ° C. are introduced into a measuring cuvette and with 0.01 ml of duodenal juice mixed.  The mixture is then quickly mixed with 0.2 ml of a 2 × 10-3 molar solution of a substrate according to the invention.  The amount of p-nitroaniline released from the substrate by the trypsin is determined by measuring the change in the optical density A OD per minute at a wavelength of 405 nm.  The amount of p-nitroaniline released per minute or, correspondingly, the change in optical density is proportional to the trypsin activity. 

  When using the substrate produced according to Example 10, Nf-Cbo-C.     Cyclohexylglycyl-Gly-Arg-pNA. HC, you can z. B.  Determine trypsin amounts down to 0.01 NF units of trypsin per ml in the test mixture. 



   Table IV below shows the activity of bovine trypsin measured by means of the substrates according to the invention at constant substrate concentration and enzyme concentration. 



   Table IV
Activity of bovine trypsin, measured by means of the substrates according to the invention with constant substrate and
Enzyme concentration.  For comparison, the corresponding with Nt-Bz-Phe-Val-Arg-pNA.     HCI and N "-Bz-DL-Arg-pNA.     HCI determined values given. 



  Substrate concentration 2 x 104 M amount of per minute by 1 NF unit
Trypsin from the substrates enzymatically released p-nitroaniline in nanomoles N "-Bz-Phe-Val-Arg-8.4 pNA. HCl N "-Bz-DL-Arg-0.45 pNA. HCI 1 20.9 II 10.8 III 28.5 IV 19.2 V 15.9 Vl 19.1 VII 25.7 VIII 14.2 IX 17.5 X 41.3 XI 33.3 XII 19.8 XIII 16.4 XIV 16.8 XV 24.5 XVI 33.9 XVII 28.1 XVIII 19.9 XIX 24.1 XX 12.4 XXI 21.0 XXII 19.9 XXIII 16.7 XXIV 15.8 XXV 21, 6 XXVI 17.5
Table IV (continued) XXVII 15.0 XXVIII 18.3 XXIX 19.1 XXX 22.4 XXXI 15.6 XXXII 18.5 XXXIII 12.8 XXXIV 14.0 XXXV 22.9 XXXVI 25.4 XXXVII 24.8 XXXVI II 22.0 XXXIX 29.1 XL 31.2
The results given in Table IV were determined under the following test conditions:

  In a cuvette, 1.7 ml of tris-imidazole buffer with a pH of 8.4 and an ionic strength of 0.30 at 37 ° C. were mixed with 0.1 ml of an aqueous trypsin solution with a concentration of 2 NF units per ml .  0.2 ml of a 2 x 10-3 molar solution of the substrate to be tested was added to the mixture. 



  Then the amount of p-nitroaniline released from the substrate by the trypsin was determined by measuring the change in the optical density A OD per minute at a wavelength of 405 nm. 



   The substrates according to the invention are also suitable for determining inhibitors of plasminogen activators, e.g. B.  of urokinase inhibitors.  This determination can be carried out by incubating a measured amount of urokinase with a body liquid containing urokinase inhibitors in the presence or absence of a buffer system, diluting the incubated mixture to a volume of 1.8 ml using tris-imidazole buffer and using the diluted mixture 0.2 ml of a 2 x 10-3 molar solution of a substrate according to the invention are added.  The uninhibited urokinase activity is determined by measuring the change in optical density per minute.  The difference between the amount of urokinase used and the remaining urokinase activity is a measure of the amount of inhibitor present. 

  Trypsini inhibitors can be determined using the same method. 



   In order to determine plasminogen preactivators in plasma, the latter are first activated quantitatively, whereupon the plasminogen activators formed in this way are determined using the substrates according to the invention.  The plasminogen preactivators are activated by optimally incubating the plasma after adding an active Hagemann factor preparation and adding an aprotinin-containing trisimidazole buffer to the incubate in order to avoid the other enzymes that also arise when the plasminogen preactivators are activated, in particular plasma kallikrein and plasmin to inhibit. 

 

   To determine trypsinogen in body fluids, an activator, e.g. B.  Chymotrypsin, added to convert the trypsinogen to trypsin, which can then be determined using the substrates according to the invention in the manner described above.  The principle of the above-described determinations by means of the substrates according to the invention consists in unrestrained enzyme which is already present or has been formed by activation or is still present after inhibition, i.e. H.  Plasminogen activator (e.g. B.  Urokinase) or trypsin.  


    

Claims (3)

 PATENT CLAIMS 1. Tripeptide derivatives of the formula R'-X-Y-Z-NH-R2 in which X is a group of the formula EMI1.1  in which R3 denotes a straight-chain or branched alkyl radical having 1 to 7 carbon atoms or a cyclohexyl or cyclohexylmethyl radical, Y represents a seryl group or a group of the formula -NH- (CH2) n-CO-, in which n is an integer from 1 to 7, Z represents an arginyl or lysyl group, R 'represents hydrogen or an acyl group and R2 represents an aromatic hydrocarbon radical which may have substituents.  Tripeptide derivatives according to claim 1, characterized in that they are mixed with a mineral acid, e.g. HCl, HBr, H2S04 or H3PO4, or an organic acid, e.g. Formic, acetic, oxalic or tartaric acid are protonized.  3. Esterolytic method (for urokinase). This method makes use of the property of urokinase to catalyze the hydrolysis of the methyl N-acetyl-L-lysine directly. This method has also to create the so-called. CTA urokinase unit served.   (CTA = Committee on Thrombolytic Agents of the National Heart Institute, USA). A CTA unit is the amount of urokinase which releases 46.2 x 10-3 pLM M methanol from Nt-acetyl-L-lysine methyl ester in 37 ° C in 1 hour (see NU Bang et al., Thrombosis and Bleeding Disorders , Georg Thieme Verlag, Stuttgart, 1971, p. 377) This method can only be used for the determination of pure urokinase preparations, but is not suitable for the determination of urokinase in body fluids or tissue extracts, since the ester mentioned contains other proteolytic enzymes ( e.g. plasmin, plasma kallikrein, etc.) is split faster than by urokinase. ** WARNING ** End of CLMS field could overlap beginning of DESC **.  3. tripeptide derivatives according to claim 1, characterized in that the acyl group R 'has the partial formula R4-CO-, in which R4 a) an aliphatic hydrocarbon radical with 1 to 17 carbon atoms, optionally bearing an amino group in the o-position, b) an araliphatic hydrocarbon radical whose aliphatic radical has 1 to 17 carbon atoms and whose aryl radical optionally carries an amino group, c) a cycloaliphatic hydrocarbon radical optionally bearing an amino or aminomethyl group, d) an aromatic hydrocarbon radical optionally bearing an alkyl, amino or aminoalkyl group or e) a benzyloxy group represents.  Tripeptide derivatives according to claim 1, characterized in that the sulfonyl group Rl is an alkanesulfonyl group with 1 to 17 carbon atoms in the alkane radical, e.g. a methane or ethanesulfonyl group, or an arylsulfonyl group optionally carrying at least one lower alkyl substituent, e.g. is a benzene, p-toluene or naphthalenesulfonyl group.  5. Tripeptide derivatives according to claim 1, characterized in that R2 is a p-nitrophenyl, naphthyl or 4-metoxy-2-naphthyl group.  The invention relates to tripeptide derivatives for the determination of plasminogen activators, their inhibitors and plasminogen preactivators as well as trypsin, trypsin inhibitors and trypsinogen.  The human organism produces several activators which convert the plasminogen proenzyme into the active plasmin lysis enzyme. This group of activators includes e.g. Tissue and blood kinase and urokinase. These activators play an important role in the mechanism of blood coagulation. If these activators are released abnormally large, there is a risk of increased fibrinolysis activity and thus an increased tendency to bleed or hemorrhage. Conversely, if these activators are released too weakly, the balance between coagulation capacity and fibrinolysis is disturbed and, as a result, there is an increased risk of thrombosis. The determination of plasminogen activators in body fluids and tissue extracts is therefore of great importance in clinical practice, e.g. from I. Witt in Biochemistry of Blood Coagulation and Fibrinolysis, Verlag Chemie, Weinheim, 1975, p. 119, is defined as follows: The determination of fibrinolytic activity in plasma or serum serves firstly to identify hyperfibrinolytic states with which various clinical pictures are associated. In addition, good results in the lysis of intravascular thrombi by administration of plasminogen activators have been achieved in recent years. To control this thrombolytic therapy, measurements of the fibrinolytic activity are also essential. F. E.. Smyrniotis et al. [Thromb. Diath. et Haemorrh. Vol. III, 257-70 (1959)] include the following: The excretion of urokinase in normal people is independent of age, gender and amount of urine. The urokinase excretion is increased after the occurrence of a myocardial infarction and after an attack of coronary insufficiency. Elimination is reduced in patients with carcinosis, cardiac congestion and uremia. These differences suggest that significant changes in the fibrinolytic system of the plasma can occur as a result of these diseases. So far, there are no really reliable methods for determining plasminogen activators in body fluids and organ extracts. In principle, three methods are known.  1. Spontaneous lysis of a blood clot. Blood is allowed to clot spontaneously or by adding thrombin and the spontaneous lysis of the clot is observed at 37 ° C. A lysis usually takes place after 24 hours. This method is non-specific since the activator activity is not direct, but indirect via the lysis enzyme plasmin is measured (see I. Witt Biochemistry of Blood Coagulation and Fibrinolysis, Verlag Chemie, Weinheim, 1975, p. 119).  2. Casein hydrolysis. This method uses the property of the plasmin to hydrolytically break down casein. Casein is incubated with the sample to be examined. At the beginning and at the end, an aliquot of the incubation mixture is mixed with trichloroacetic acid. In the supernatant, the tyrosine content is determined by spectrophotometric measurement at 280 nm, from which the activator effectiveness can be approximately calculated indirectly [L. F. Remmert et al., J. Biol. Chem. 181, 431 (1949)].