CH609154A5 - Method for the quantitative determination of proteolytic enzymes in body fluids from humans and mammals - Google Patents

Abstract

Method for the quantitative determination of proteolytic enzymes of group E.C.3.4.4., especially plasma kallikrein, in body fluids of humans and mammals comprises allowing a substrate to act on the body fluid, which substrate has the following structure R<1>-Pro-X-Arg-NH-R<2>, in which R<1> is an acyl or sulphonyl group, R<2> is an aromatic hydrocarbon radical which optionally carries substituents, and X is a phenylalanyl, beta -cyclohexylalanyl, phenylglycyl or tyrosyl group, where -NH-R<2> is a chromophoric group, and which has the property of liberating, under the hydrolytic action of the said enzymes, a cleavage product of the formula NH2-R<2> and comprises measuring the amount of the product NH2-R<2> which has been cleaved off by the enzyme using photometric, spectrophotometric or fluorescence photometric methods.

Description

  

  
 

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

 



   PATENT CLAIMS
1. Method for the quantitative determination of proteolytic enzymes of the classification group E.C. 3.4.4. in body fluids of humans and mammals, characterized in that a body is allowed to act on the body fluid, which has the following structure:

  : R '-Pro-X-Arg-NH-R2 I in which Rl is an acyl or sulfonyl group, R2 is an aromatic hydrocarbon radical which may have substituents and X is phenylalanyl, ss-cyclohexylalanyl; Denote phenylglycyl or tyrosyl group, where -NH-R2 represents a chromophoric group, and which has the property, under the hydrolytic action of the enzymes mentioned, a cleavage product of the formula
NH2-R2 and that the amount of the product NH2-R2 cleaved by the enzyme is measured by means of photometric, spectrophotometric or fluorescence-photometric methods.



   2. The method according to claim 1, characterized in that one uses a substrate whose acyl group denoted by Rl has the following partial formula: R3 - CO - II, wherein R3 a) an aliphatic hydrocarbon radical with 1 to 17 carbon atoms, b) an araliphatic hydrocarbon radical, the aliphatic part of which contains 1 to 17 carbon atoms and the aryl radical optionally carries an amino group, c) a cycloaliphatic hydrocarbon radical optionally bearing an amino or aminomethyl group, d) an optionally an alkyl, amino or aromatic hydrocarbon radical bearing aminoalkyl group or e) denotes a benzyloxy group.



   3. The method according to claim 1, characterized in that one uses a substrate whose sulfonyl group denoted by R 'is a benzenesulfonyl or p-toluenesulfonyl group.



   4. The method according to claim 1, characterized in that one uses a substrate in which R2 is a p-nitrophenyl, p-naphthyl or 4-methoxy-p-naphthyl group.



   5. The method according to claim 1, characterized in that blood plasma is used as body fluid.



   6. A method for determining plasma kallikrein according to claims 1 and 5.



   7. The method according to claim 1, characterized in that the cleavage product NH2R2 is diazotized before the photometric measurement and coupled with a coupling component.



   8. The method according to claim 1, characterized in that the substrate is used in protonated form.



   9. The method according to claims 1 and 8, characterized in that the substrate is used in the form of the hydrochloride.



   The present invention relates to a method for the quantitative determination of proteolytic enzymes of the classification group E.C. 3.4.4. in body fluids of humans and mammals, in particular for the determination of prekallikrein, prekallikrein activators, kallikrein and kallikrein inhibitors in blood plasma.



   There are currently various synthetic products as substrates for proteolytic enzymes of the peptide-peptidohydrolase group, in particular those enzymes which cleave the peptide chains on the carboxyl side of arginine and lysine, e.g. B. trypsin, thrombin, plasmin and kallikrein and other class E.C. 3.4.4 (Enzyme gymnastics). E.C. is the abbreviation for Enzyme Committee of the International Union of Biochemistry.



   According to the type of catalytic reaction that the proteolytic enzymes trigger, i. H. the esterolytic or the amidolytic reaction, the synthetic substrates mentioned can be divided into two main groups, namely the group of the ester substrates and the group of the amide substrates.



   The ester substrates have hitherto been used far more frequently than the amide substrates for the investigation of reactions in which the enzymes mentioned take part, because the ester substrates are cleaved faster than the currently available amide substrates.



   Work has been published (see, for example, BWH SEE GERS, Blood Clotting Enzymology, Academic Press, 1967, pp. 36 and 42-44), from which it emerges that the ratio of the reaction rates of the esterolytic and amidolytic catalysis of the enzymes mentioned fluctuates widely is subject. For example, acetylated thrombin has an increased esterolytic activity, while amidolytic activity is almost eliminated. In biological fluids, the esterolytic activity of the enzymes mentioned using low molecular weight ester substrates is not always inhibited to the same extent as the amidolytic activity determined using natural substrates or amide substrates.



  Using a synthetic chromogenic amide substrate with a structure derived from the structure of the natural substrates, it would be easier to determine the increase or decrease in the enzymatic activity in biological fluids.



   Natural substrates, such as kininogen and fibrinogen, which usually have high molecular weights, are very difficult to isolate from natural sources in pure native form.



  The enzymatic hydrolysis of these natural substrates gives rise to products which can be prepared using customary methods, e.g. B. by spectrophotometric measurements, difficult to determine and track.



   The object underlying the present invention was to develop a synthetic amide substrate which should be easily and quickly hydrolytically cleavable by plasma kallikrein and which should provide cleavage products which should be easy to determine by photospectrometric methods.



   To solve this problem, it was assumed that kallikrein in shock states in humans by hydrolytic action from the naturally occurring substrate kininogen (molecular weight about 50,000) releases the biologically highly effective, pain-producing bradykinin (molecular weight about 1000) and that by using a C -terminal structural element of bradykinin and linking this element with a chromogenic or fluorescent group could receive a synthetic amide substrate which has the same or approximately the same susceptibility to plasma kallikrein as the natural substrate and which is chromolytically or amidolysed

   would form fluorescent fission product with a high molar extinction coefficient measurable at high wavelengths, so that the influence of biological liquids on the measurement would be greatly reduced.



   It has now become a synthetic chromogenic or fluores



  decorative substrate that meets the requirements defined above and for the determination of proteolytic enzymes of the classification group E.C. 3.4.4. in human and mammalian body fluids, by photometric, spectrophotometric or fluorescence-photometric methods. This substrate has the general formula R '-Pro-X-Arg-NH-R2 I, in which R' is an acyl or sulfonyl group, R2 is an aromatic hydrocarbon radical which may have substituents, X is phenylalanyl- "ss-cyclohexylalanyl; phenylgly denote cyl or tyrosyl group, where -NH-R2 represents a chromophore group, and has the property, under the hydrolytic action of the enzymes, to release a cleavage product of the formula NH2-R2, the amount of which can be measured by the photometric methods mentioned.



   The acyl group denoted by R 'in formula I can be characterized by the following group formula:
R3-CO wherein R3 (a) an aliphatic hydrocarbon residue with 1 to 17 carbon atoms, optionally bearing an amino group, (b) an araliphatic hydrocarbon residue, the aliphatic part of which contains 1 to 17 carbon atoms and the aryl residue optionally contains an amino group bears, (c) a cycloaliphatic hydrocarbon residue optionally bearing an amino or aminomethyl group, (d) an optionally an alkyl, amino or
Aromatic hydrocarbon group bearing aminoalkyl group or (e) denotes a benzyloxy group.



   R3 can in particular be an alkyl group, such as methyl, ethyl,
Propyl, butyl, pentyl, etc. to heptadecyl. These alkyl groups can carry an amino group in the co-position. Thus R3 can e.g. B. be an o-aminopropyl, c3-aminopentyl or o-ami nononyl group. R3 may further denote a benzyl, 2-phenylethyl, 3-phenylpropyl, etc. to 1 1-phenylundecyl group. The phenyl radical of the groups mentioned can carry an amino group in the p-position. Thus, R3 can be a p-aminobenzyl, 2- (p-aminophenyl> ethyl, 3Xp-aminophenylS propyl, etc. to 1 lXp-aminophenyl-undecyl group.

  R3 can also represent a cyclohexyl, 4-aminocyclohexyl, 4-amino methylcyclohexyl, 4-aminoethylcyclohexyl, 4-aminopropyl cyclohexyl or 4-aminobutylcyclohexyl group.



   R3 can also be a phenyl, a-naphthyl, ss-naphthyl or
Represent 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 can e.g. B. methyl, ethyl, propyl, isopropyl, butyl or isobutyl. Finally, R3 can represent a benzyloxy group.



   The sulfonyl group denoted by R1 in formula I can be a benzenesulfonyl or p-toluenesulfonyl group.



   The preparation of the substrate can be done according to various, e.g. T. known methods take place:
1. In the first method, the chromophoric groups (R2 in formula I) are attached to the C-terminal amino acid group. These groups simultaneously exercise the function of
C-terminal carboxyl protecting groups during the stepwise connection of amino acids to the desired peptide structure. The remaining protective groups are selectively split off from the final product without the chromophores
Group is influenced. This method is e.g. B. in peptides
Synthesis by Miklos Bodanszky et al., Interscience Publis hers, 1966, pages 163-165.



   2. In the second method, the chromophoric group is coupled to the finished peptide structure, namely that after the stepwise 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 chromophoric group is attached to the Carboxyl group is attached. The remaining protective groups are finally split off selectively, under conditions in which the chromophoric group remains intact. This method is described in Peptide Synthesis, see above, pages 43 and 44.



   3. In the third method, the chromophoric group is coupled to the finished peptide structure after the remaining protective groups have been removed.



  The C-terminal carboxyl group is released in this case by racemization-free enzymatic ester cleavage.



  These ester-splitting enzymes can either be free or bound to a matrix.



   To protect the Na amino groups during the step-by-step construction of the peptide chains, ordinary, known amino protecting groups which can be cleaved off selectively can be used. It’s 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 a methyl, ethyl or isopropyl ester during the step-by-step construction of the desired peptide structure.



   The remaining free reactive groups which are not involved in the construction of the peptide structure can be blocked by the following special measures: The ô-guanidine group of arginine is protected by NO2, Tos or by simple protonization.



   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 developed 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).

 

   The following abbreviations are used in the present description (including patent claims): Arg = arginine Pro = proline Phe = phenylalanine Tyr = tyrosine Val = valine Ph = C-phenylglycine CHA = B-cyclohexylalanine
Unless otherwise noted, all amino acids in the peptide chains described in the examples have an L configuration.



   Ac = Acetyl AclO = Acetic anhydride AcOH = Acetic acid BOC = Tert-butyloxycarbonyl Bz = Benzoyl Bzl = Benzyl BzzO = Benzoic anhydride Cbo = Carbobenzoxy DCCI = Dicyclohexylcarbodiimide DCHA = Dicyclohexylamine DCH = Dicyclohexylethylaminomethane Lmethylamine DMC layer

  Chloroform / methanol 9: 1 LMS B = solvent system: n-propanol / methyl acetate / water 7: 1: 2 LMS C = solvent system: n-butanol / acetic acid / water 3: 1: 1 HMPTA = N, N, N: N ' , N ", N" -hexamethylphosphoric acid triam MCbo = p-methoxyphenylazocarbobenzoxy MeOH = methanol NA = naphthylamine OtBu = tert-butyloxy OEt = ethyloxy OMe = methyloxy OpNP = p-nitrophenoxy OisoPr = iso-propyloxy pNA = p-nitroanifluoro TFA Tetrahydrofuron Tos = p-toluenesulfonyl Example 1 N -Bz-Pro-Phe-Arg-p NA.HCl Ia) N -Cb: Arg (NO2) -p NA
1.

  32.55 g (92.1 mmol) of well-dried Cbo-Arg (NO2-OH in 200 ml of N, N, N ', N: N ", N" - dried and freshly distilled over P205) were placed in a 500 ml three-necked round bottom flask. Hexamethylphosphoräsuretriamid dissolved with exclusion of moisture at 20 C. To this solution were first added 9.32 g (92.1 mmol) of Et3N and then 18.90 g (115.1 mmol) of p-nitrophenyl isocyanate in excess of 250 / above after 24 hours Reaction time 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 each of 20% NaHCO3 solution above, three times with 0.7 liter dist.

  Water, three times with 0.5 liters of 0.5N HCL and finally three times with 0.5 liters of dest. 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 -cbo- (o-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 after drying twice with 50 ml of DMF heated to 70 ° C., the desired product being completely dissolved out, while the by-product, namely N, N'-bis-p-nitrophenylurea, The DMF solution was concentrated in vacuo at 40 ° C.

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



   Yield: 29.75 g (68.2 / o of theory). The following values were determined by elementary analysis and calculation from the gross formula C20H23N7C7 (the values for the gross formula are in brackets):
C = 50.42 / o (50.74 / 0); H = 4.98% (4.9001o); N = 20.90% (20.710 / o). [a] ss2 = 1.27 (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.degree. C., 6.85 g (50 mmol) of isobutyl chloroformate, dissolved in 30 ml of THF, were then added dropwise over a period of 15 minutes, maintaining a temperature of -10 to -5.degree. 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, digested three times with 50% NaHCO3 solution above and again three times with distilled water. After drying in vacuo, the crude product was dissolved in methanol and passed over a methanol equilibrated column of Sephadex LH-20 (cross-linked dextran gel). A fraction of the eluate gave 7.67 g of substance (32.40 / o 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) of DCCI and 8.2 g (50 mmol) of 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 column of Sephadex LH-20 equilibrated with MeOH. Besides a large number of by-products, namely N-Cbo-Arg- (NO2) -N, N '-dicyclUhexylurarn, 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 end product prepared according to paragraph la.



  Ib) Na-Cbo-Phe-Arg (N02) p NA
With exclusion of moisture, 9.5 g (20 mmol) of N-Cbo-Arg (NO2) -p NA (see Example 1, paragraph la) were stirred in a flask with 80 ml of 2-n HBr in glacial acetic acid for one hour at 20 ° C. The reaction solution was slowly added dropwise to 400 ml of dried ether while stirring vigorously, with HBr-H-Arg (NO2) -p-NA precipitating out, and 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 and excess HBr and acetic acid formed.

  After drying in vacuo over NaOH platelets, the deblocked product was obtained in quantitative yield. The dry hydrobromide derivative was dissolved in 50 ml of DMF, cooled to -10 ° C., and 4.16 ml (30 mmol) of Et3N were added to release H-Arg (NO2) -p NA from the hydrobromide. The EtsNHBr- Salt was filtered off, washed with a little cold DMF, and 7.8 g (21 mmol) of Cbo-Phe-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 -10 ° C. and buffered with 1.4 ml (10 mmol) Et3N. After about 5 hours another 1.4 ml Et3N was added.

  After a further 24 hours, the reaction solution was evaporated to dryness in vacuo at 40 ° C. The residue was digested three times with 100 ml of distilled H20 each time and again dried in vacuo at 40 ° C. over NaOH plates. The dried product was recrystallized from methanol, whereby 7.84 g of substance which was chromatographically uniform in the solvent systems A and B were obtained. A further 3.14 g of substance were obtained from the mother liquor by gel filtration through a column of Sephadex LH-20 equilibrated with MeOH.



  In total, 10.98 g (88.5% of theory) became more uniform
Received substance with a mp. Of 203-205 C. Elemental analysis and calculation from the gross formula C29H32N808 gave the following values (the values for the gross formula are given in brackets): C = 55.88% (C = 56.12%); H = 5.050 / 0 (H = 5.20%); N = 18.31% (N = 18.060 / o).



      Ic) N -Cbo-Pro-Phe-Arg (NO2) -p NA
31.0 g (50 mmol) of N -Cbo-Phe-Arg (NO2) -p NA (see Example 1, Section Ib) were mixed with 250 ml of 2-n HBr in glacial acetic acid (0.5
Mol) treated and worked up as in Example lb). The above
NaOH plates vacuum dried hydrobromide
Dipeptide derivatives were dissolved in 150 ml DMF and, after cooling, 7.6 g Et3N in 15 ml DMF (75 mmol) were added. Formed Et3N.HBr was filtered off and washed with a little cold DMF. 18.5 g (50 mmol) of Cbo-Pro were added to the filtrate
Given OpNP. Further processing was carried out analogously to Example 1, paragraph Ib.

  By gel filtration on one with
MeOH-equilibrated Sephadex LH-20 columns were made after
Elution with MeOH 30.9 g (86.2% of theory) from in the
Solvent systems A and B chromatographically uniform substance with a mp. 184-186 C obtained. The elementary analysis and the calculation from the gross formula C34H3sNsOs gave the following values: C = 56.750 / o (56.900 / o); H = 5,500 / o (5.48%); N = 17.700 / c (17.560 / o) (the values for the
Gross formulas are in parentheses).



   Id) N -Bz-Pro-Phe-Arg (NO2} p NA
14.4 g (20 mmol) Na-Cbo-Pro-Phe-Arg (NO2} p NA (see Example 1. Paragraph Ic) were treated with 120 ml 2-n HBr in glacial acetic acid (0.24 mol) was worked up as in Example 1, paragraph Ib).



   After the dried tripeptide hydrobromide derivative had been dissolved in 60 ml of DMF, 4.16 ml of Et3N (30 mmol) were added to the solution after cooling. The Et3N-HBr formed was filtered off and washed with a little cold DMF. 6.8 g (30 mmol) of benzoic anhydride were added to the filtrate obtained. It was then buffered and worked up as in Example 1, paragraph Ib). The crude product obtained was purified over a SeHadex LH-20 column equilibrated with MeOH. In this way, 11.3 g (82.00 / o of theory) of amorphous substance which was chromatographically uniform in the solvent systems A and B were obtained. The elementary analysis and the calculation from the gross formula C33H37N908 gave the following values: C = 57.51% (57.63%); H = 5.38% (5.42%); N = 18.47% (18.33%).



  Ie) Na-Bz-Pro-Phe-Arg-p NA HCl
688 mg (1 mmol) of N -Cbo-Pro-Phe-Arg (NO2} p NA (see Example 1, Section Id) were weighed into the reaction vessel of a Sakakibara apparatus, and 10 ml of dried hydrogen fluoride gas were condensed in the reaction vessel The nitro protective group was removed with stirring for 1 hour at 0 C. 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, 0.5 ml of concentrated HCl were added and the solution After repeating this process twice, the residue was dissolved in 50 ml of 30% acetic acid.

  For cleaning, the acetic acid solution was applied to a Sephadex G-15 column equilibrated with 300% acetic acid and eluted with 30% acetic acid. After freeze-drying part of the eluate, 572 mg (84.2% of theory) of an amorphous powder were obtained, which was uniform in the solvent system C by thin layer chromatography. Elemental analysis and calculation from the gross formula G3H39N806CI gave the following values: C = 58.12% (58.36%); H = 5.70% (5.79%); N = 16.65% (16.50%) and Cl = 5.15% (5.22%).



   The amino acid analysis showed the expected amino acids in the correct ratio:
Arg: 0.98 - Phe: 1.0 - Pro: 0.97
Example 2
II. N -Ac-Pro-Phe-Arg-pNA HCl
IIa) N "-Ac-Pro-Phe-Arg (NO2> pNA
1.44 g (2 mmol) of the compound prepared according to Example 1, paragraph Ic, was deblocked according to Example 1, paragraph Ib, dissolved in 10 ml DMF and mixed with 420 l (3 mmol) Et3N. After cooling, the reaction mixture was mixed with 400 l (4 4 mmol) of Ac2O were added and further treated according to Example 1, paragraph Id. Purification: Gel filtration on Sephadex LH-20 in methanol. Yield: 1.05 g of amorphous substance (83.9% of theory); uniform in the DSC in LMS A and B.



  Elemental analysis and calculation from the gross formula C28H35N9O8 gave the following values: C = 53.68% (53.75O / O); H = 5.60% (5.64%); N = 20.20% (20.15%).



  II. Na-Ac-Pro-Phe-Arg-pNA HCl
626 mg (1 mmol) of compound IIa were converted to compound II according to Example 1, paragraph le. Purification: Gel filtration on Sephadex G-15 in 30/0 AcOH. Yield: 521 mg of freeze-dried amorphous powder (84.4% of theory); uniform in the DSC in the LMS C. Elementary analysis and calculation from the gross formula C28H37N8O6Cl gave the following values: C = 54.25% (54.500 / o); H = 6.01% (6.04%); N =
18.25% (18.16%); Cl = 5.71% (5.750 / o).



   The amino acid analysis showed the expected amino acids in the correct ratio: Arg: 0.99 - Phe: 1.00 - Pro: 0.97.



  Example 3 III. Na-Capryloyl-Pro-Phe-Arg-pNA HCl Illa) Na-Capryloyl-Pro-Phe-Arg (NO2} pNA 718mg (1 mmol) of the compound prepared according to Example 1, paragraph Ic, were prepared according to Example 1, paragraph Ib, deblocked, dissolved in 10 ml of DMF and mixed with 210 ul (1.5 mmol) of Et3N. After cooling, 400 mg (1.5 mmol) of caprylic acid p-nitrophenyl ester were added to the reaction mixture and the reaction product according to Example 1, paragraph Ib, Purification: Gel filtration on Sephadex LH-20 in methanol. Yield: 490 mg amorphous substance (68.8 / o of theory), uniform in the DSC in LMS A and B.



  Elemental analysis and calculation from the gross formula C34H49N9O8 gave the following values: C = 57.51% (57.37%); H = 6.88% (6.94%); N = 17.80% (17.71%).

 

     III. Na-capryloyl-Pro-Phe-Arg-pNA HCl
356 mg (0.5 mmol) of compound III were converted to compound III according to Example 1, paragraph Ie. Purification: Gel filtration on Sephadex G-15 in 30% AcOH. Yield: 226 mg of freeze-dried amorphous powder (64.3% of theory); uniform in the DSC in the LMS C.



   Elemental analysis and calculation from the gross formula C34H51N8O6Cl gave the following values: C = 57.920 / 0 (58.07%); H = 7.18% (7.31%); N = 16.08% (15.93%); Cl = 4.98% (5.04%).



   The amino acid analysis showed the expected amino acids in the correct ratio: Arg: 0.99 - Phe: 1.00 - Pro: 0.95 Example 4 IV. Nα-Cyclohexylcarbonyl-Pro-Phe-Arg-pNA.HCl IVa) Nα; -Cyclohexylcarbonyl-Pro-Phe-Arg (NO2) -pNA
718 mg (1 mmol) of the compound obtained according to Example 1, paragraph Ic, was deblocked according to Example 1, paragraph Ib, dissolved in 10 ml DMF and mixed with 210 l (1.5 mmol) Et3N. After cooling, the reaction mixture was 375 mg (1.5 mmol) p-nitrophenyl cyclohexane carboxylate added. The reaction product was treated according to Example 1, paragraph Ib. Purification: Gel filtration on Sephadex LH-20 in methanol. Yield: 593 mg amorphous substance (85.5% of theory); uniform in the DSC in LMS A and B.



   Elemental analysis and calculation from the gross formula C33H43N9O8 gave the following values: C = 57.01% (57.13%); H = 6.18% (6.25%); N = 18.300 / 0 (18.170 / 0).



  V. Nα-Cyclohexylcarbonyl-Pro-Phe-Arg-pNA.HCl
347 mg (0.5 mmol) of compound Va were converted to compound V according to Example 1, paragraph Ie. Purification: Gel filtration on Sephadex G-15 in 30 / O AcOH. Yield: 295 mg of freeze-dried amorphous powder (86.10 / or theory); uniform in the DSC in the LMS C.



   Elemental analysis and calculation from the gross formula C33H4sNsO6CI gave the following values: C = 57.70% (57.84%); H = 6.66 / o (6.62%); N = 16.40% (16.35%); Cl = 5.110 / 0 (5.17%).



   The amino acid analysis showed the expected amino acids in the correct ratio: Arg: 0.97 - Phe: 1.00 - Pro: 0.95.



  Example 5 V. N? -4-Methyl-benzoyl-Pro-Phe-Arg-pNA.HCl Va) N? -4-Methyl-benzoyl-Pro-Phe-Arg (NO2) -pNA
718 mg (1 mmol) of the compound prepared according to Example 1, paragraph Ic were deblocked according to Example 1, paragraph Ib, dissolved in 10 ml DMF and mixed with 210 l (1.5 mmol) Et3N. After cooling, 386 mg (1.5 mmol) of p-toluenesulfonic acid p-nitrophenyl ester were added to the reaction mixture. The reaction product was treated according to Example 1, paragraph Ib. Purification: Gel filtration on Sephadex LH-20 in methanol. Yield: 591 mg amorphous substance (84.2% of theory); uniform in the DSC in LMS A and B.



   Elemental analysis and calculation from the gross formula C34H39N9O8 gave the following values: C = 58.070 / 0 (58.190 / 0); H = 5.530 / 0 (5.600 / 0); N = 18.06% (17.97%).



  V. N? -4-Methyl-benzoyl-Pro-Phe-Arg-pNA.HCl
351 mg (0.5 mmol of the compound Va were converted to the compound V according to Example 1, paragraph le) Purification: Gel filtration on Sephadex G-15 in 300/0 AcOH. Yield: 248 mg of freeze-dried amorphous powder (71.5% of theory ); uniform in the DSC in LMS C.



   Elemental analysis and calculation from the gross formula C34H4lNsO6Cl gave the following values: C = 59.01% (58.910 / 0); H = 5.86% (5.96%); N = 16.31 / o (16.17%); Cl = 5.07% (5.110 / 0).



   The amino acid analysis showed the expected amino acids in the correct ratio: Arg: 0.96 - Phe: 1.00 - Pro: 0.96.



  Example 6 Vla) N? -3-phenyl-propionyl-Pro-Phe-Arg-pNA.HCl Vla? N? -3-phenyl-propionyl-Pro-Phe-Arg (NO2) -pNA
718 mg (1 mmol) of the compound obtained according to Example 1, paragraph Ic, were added according to the example. After cooling, 407 mg 1, paragraph Ib, are unblocked, dissolved in 10 ml DMF and added with 210111 (1.5 mmol) Et3N (1.5 mmol) p-nitrophenyl dihydrocinnamate. The reaction product was carried out according to Example 1, paragraph Ib , further treated cleaning: gel filtration on Sephadex LH-20 in methanol. Yield: 547 mg amorphous substance (76.4% of theory); uniform in the DSC in LMS A and B.



   Elemental analysis and calculation from the gross formula C35H41N0O8 gave the following values: C = 58.58% (58.730 />); H = 5.69% (5.77%); N = 18.82% (17.61%).



  VI. N? -3-phenyl-propionyl-Pro-Phe-Arg-pNA.HCl
358 mg (0.5 mmol) of compound VIa were converted to compound VI according to Example 1, paragraph le. Cleaning: Gel filtration on Sephadex G-15 in 300 /> AcOH. Yield: 303 mg of freeze-dried amorphous powder (85.7% of theory); uniform in the DSC in the LMS C.



   Elemental analysis and calculation from the gross formula C35H43N8O6Cl gave the following values: C = 59.24% (59.440 />); H = 6.11% (6.13%); N = 15.98% (15.85%); Cl = 4.93% (5.01%).



   The amino acid analysis showed the expected amino acids in the correct ratio: Arg: 0.98 - Phe: 1.00 - Pro: 1.01.



  Example 7 VIIa) Nα - (# - Aminocaproyl) -Pro-Phe-Arg-pNA.2 HCl VIIa Na- (NW-Cbo-Aminocaproyl-Pro-Phe-Arg (N02)
718 mg (1 mmol) of the compound prepared according to Example 1, paragraph Ic, were deblocked according to Example 1, paragraph Ib, dissolved in 10 ml DMF and mixed with 210 μl (1.5 mmol) Et3N. After cooling, 490 mg (1.25 mmol) of NCbo-o-aminocapronic acid p-nitrophenyl ester were added to the reaction mixture. The reaction product was treated according to Example 1, paragraph Ib. Purification: Gel filtration on Sephadex LH-20 in methanol. Yield: 698 mg amorphous substance (84.0% of theory); uniform in the DSC in LMS A and B.



   Elemental analysis and calculation from the gross form 40H50N10O10 gave the following values: C = 57.68% (57.82%): H = 6.01% (6.07%); N = 17.02% (16.86%).



     VII. Na- (o-aminocaproyl) -Pro-Phe-Arg-pNA-2 HCl
415 mg (0.5 mmol) of the compound VIIa were converted to the compound VII in accordance with Example 1, paragraph le. Purification: Gel filtration on Sephadex G-15 in 30% AcOH. Yield: 247 mg of freeze-dried amorphous powder (68.2% of theory); uniform in the DSC in the LMS C.



   Elemental analysis and calculation from the gross formula C32H47N906Ch gave the following values: C = 52.85% (53.04 / O); H = 6.50% (6.54%); N = 17.53% (17.40%); Cl = 9.69% (9.79%).



   The amino acid analysis gave the expected amino acids in the correct ratio: Arg: 0.98 - Phe: 1.00 - Pro: 0.95.

 

  Example 8 VIIIa) N? -4-Aminomethyl-cyclohexylcarbonyl-Pro-Phe-Arg-pNA 2 HCl Viola N? -4-Cbo-Aminomethyl-cyclohexylcarbonyl-Pro-Phe-Arg (NO2> pNA
718 mg (1 mmol) of the compound prepared according to Example 1, paragraph Ic, were deblocked according to Example 1, paragraph Ib, dissolved in 10 ml DMF and mixed with 210 l (1.5 mmol) Et3N. After cooling, 455 mg (1.1 mmol) of 4-Cbo-aminomethyl-cyclohexanecarboxylic acid p-nitrophenyl ester were added to the reaction mixture. The reaction product was treated according to Example 1, paragraph Ib. Purification: Gel filtration on Sephadex LH-20 in methanol. Yield: 762 mg amorphous substance (88.9% of theory); uniform in the DSC in LMS A and B.



   Elemental analysis and calculation from the gross formula C42H52N10O10 gave the following values: C = 58.62% (58.87%); H = 6.05% (6.12%); N = 16.54% (16.35%).



     VIII. Na-4-aminomethyl-cyclohexylcarbonyl-Pro-Phe-Arg pNA-2 HCl
428 mg (0.5 mmol) of compound VIII were converted to compound VIII according to Example 1, paragraph le. Purification: Gel filtration on Sephadex G-15 in 30% AcOH. Yield: 198 mg of freeze-dried amorphous powder (52.7% of theory); uniform in the DSC in the LMS C.



   Elemental analysis and calculation from the gross formula C34H49N9O6Cl gave the following values: C = 54.22% (54.40%}; H = 6.530 /> (6.58%); N = 16.74% (16.58%); Cl = 9.35% (9.45%).



   The amino acid analysis showed the expected amino acids in the correct ratio: Arg: 0.99 - Phe: 1.00 - Pro: 1.01.



  Example 9 IX. Na-Tos-Pro-Phe-ArgpNA HCl IXa) Na-Tos-Pro-Phe-Arg (NO2SpNA
718 mg (1 mmol) of the compound prepared according to Example 1, paragraph Ic, were deblocked according to Example 1, paragraph Ib, dissolved in 10 ml DMF and mixed with 210 l (1.5 mmol) Et3N. After cooling, 380 mg (2 mmol) of tosyl chloride were added to the reaction mixture. The reaction product was further treated in accordance with Example 1, paragraph Id.



  Purification: Gel filtration on Sephadex LH-20 in methanol.



  Yield: 225 mg amorphous substance (30.5% of theory); uniform in the DSC in LMS A and B.



   Elemental analysis and calculation from the gross formula G3H39NgOgS gave the following values: C = 53.590 /> (53.72%); H = 5.31% (5.33%); N = 17.29% (17.09% ?; S = 4.15% (4.35%).



  IX. Na-Tos-Pro-Phe-Arg-pNA HCl
185 mg (0.25 mmol) of the compound IXa were converted to the compound IX according to Example 1, paragraph le. Purification: gel filtration on Sephadex G-15 in 30% AcOH. Yield: 127 mg of freeze-dried amorphous powder (69.6% of theory); uniform in the DSC in the LMS C.



   Elemental analysis and calculation from the gross formula C23H41N0OClS gave the following values: C = 54.18% (54.35%); H = 5.63% (5.67%); N = 15.52% (15.37%); Cl = 4.80% (4.86%); S = 4.44% (4.40).



   The amino acid analysis showed the expected amino acids in the correct ratio: Arg: 0.95 - Phe: 1.00 - Pro: 0.97.



  Example 10 X. N? -4-Aminophenyl-acetyl-Pro-Phe-Arg-pNA.2 HCl Xa) N? -4-Cbo-Aminophenyl-acetyl-Pro-Phe-Arg (NO2) -pNA
718 mg (1 mmol) of the compound prepared according to Example 1, paragraph Ic, were deblocked according to Example 1, paragraph Ib, dissolved in 10 ml DMF and mixed with 210 l (1.5 mmol) Et3N. After cooling, 508 mg (1.25 mmol) of 4-Cbo-aminophenyl-acetic acid pnitrophenyl ester were added. The reaction product was treated according to Example 1, paragraph Ib. Purification: Gel filtration on Sephadex LH-20 in methanol. Yield: 595 mg amorphous substance (69.9% of theory); uniform in the DSC in LMS A and B.



   Elemental analysis and calculation from the gross formula C42H43N10O10 gave the following values: C = 58.96% (59.29%); H = 5.38% (5.45%); N = 16.55% (16.460 />).



  X. N? -4-Aminophenyl-acetyl-Pro-Phe-Arg-pNA.2 HCl
425 mg (0.5 mmol) of compound Xa were converted to compound X according to Example 1, paragraph Ie. Purification: Gel filtration on Sephadex G-15 in 30% AcOH. Yield: 315 mg of freeze-dried amorphous powder (84.6% of theory); uniform in the DSC in the LMS C.



   Elemental analysis and calculation from the gross formula C34H43N9O6Cl2 gave the following values: C = 54.72% (54.84%); H = 5.80 / o (5.82%); N = 17.08% (16.93%); Cl = 9.43% (9.52%).



   The amino acid analysis showed the expected amino acids in the correct ratio: Arg: 0.99-Phe: 1.00 - Pro: 0.96.



  Example 11 XI. N? -4-aminobenzoyl-Pro-Phe-Arg-pNA.2HCl XIa) N? -4-Cbo-Aminobenzoyl-Pro-Phe-Arg (NO2) -pNA
718 mg (1 mmol) of the compound prepared according to Example 1, paragraph Ic, was deblocked according to Example 1, paragraph Ib, dissolved in 10 ml DMF and mixed with 210 µ1 mmol) Et3N. After cooling, 490 mg (1.25 mmol) of p-nitrophenyl 4-Cbo-aminobenzoate were added to the reaction mixture. The reaction product was treated according to Example 1, paragraph Ib. Purification: Gel filtration on Sephadex LH-20 in methanol. Yield: 693 mg amorphous substance (82.8% of theory); uniform in the DSC in LMS A and B.



   Elemental analysis and calculation from the gross formula C41H44N10O10 gave the following values: C = 58.92% (58.84%); H = 5.16% (5.30%); N = 16.93% (16.74%).



  XI. Na-4-aminobenzoyl-Pro-Ph e-Arg-pNA2 HCl
418 mg (0.5 mmol) of the compound Xla were converted to the compound Xl according to Example 1, paragraph le. Purification: gel filtration on Sephadex G-15 in 30% AcOH. Yield: 282 mg of freeze-dried amorphous powder (77.2% of theory); uniform in the DSC in the LMS C.



   Elemental analysis and calculation from the gross formula C33H4lNOO6Cl2 gave the following values: C = 54.09% (54.25%); H = 5,600 /> (5.66%); N = 17.480 / 0 (17.250 / 0); Cl = 9.65% (9.71%).



   The amino acid analysis showed the expected amino acids in the correct ratio: Arg: 0.95 - Phe: 1.00 - Pro: 0.97.



  Example 12 XII. Na-Cbo-Pro-Phe-Arg-pNA HCl XIIa) Cbo-Arg-pNA HCl
In a three-necked round-bottomed flask of 250 ml content, 16.0 g (47.0 mmol) of Cbo-Arg OH.HCl dried in vacuo over P2O5 were dissolved in 90 ml of absolute HMPTA with exclusion of moisture at 20 ° C. At room temperature, a solution of 4.74 g (47.0 mmol) of Et3 N in 10 ml of HMPTA and then 16.4 g (100 mmol) of p-nitrophenyl isocyanate (1,000% excess) were added in portions to the solution obtained For 24 hours at 20 C, the HMPTA was largely distilled off in vacuo. The residue was extracted several times with 30% acetic acid. The residue was discarded.

  The combined acetic acid extracts were further purified by gel filtration on a Sephadex G-15 column, as described in Example 1, paragraph Ie. After freeze-drying part of the eluate, 12.6 g of an amorphous powder were obtained, which was uniform in the DSC in the LMS C and was cleaved by trypsin to release pNA. Elemental analysis and calculation from the gross formula C20H2sN6OsCI gave the following values: C = 51.29% (51.67%); H = 5.48% (5.42%); N = 17.92% (18.08%); Cl = 7.50% (7.63%).



  XIIb) N -Cob-Phe-ArgpNA-HCl
With exclusion of moisture, 7.0 g (15 mmol) of the compound XIIa according to Example 1, paragraph Ib, were unblocked and worked up. The product thus obtained was dissolved in 75 ml of DMF, a solution of 1.52 g of Et3N in 10 ml of DMF was added after cooling, and the Et3N HBr formed was filtered off. 8.4 g (20 nmol) of Cbo-Phe-OpNP were added to the filtrate. The reaction was otherwise carried out according to Example 1, paragraph Ib. The reaction solution was evaporated to dryness in vacuo at 40 ° C., whereupon the residue was dissolved in 250 ml of MeOH and the solution was concentrated with 1.20 ml.



  Hydrochloric acid was added. The crude product was purified by gel filtration on a Sephadex LH-20 column in MeOH. Concentration in vacuo gave 7.55 g (82.2% of theory) of the desired product from a fraction, which was uniform in the DSC in the LMS C. Elemental analysis and calculation from the gross formula GgH34N706CI gave the following values: C = 57.09% (56.91%); H = 5.500 /> (5.60%); N = 16.25% (16.02%); Cl = 5.71% (5.79%).



     XII. Na-Cbo-Pro-Phe-Arg-pNA HCl
673 mg (1.1 mmol of compound Xllb were deblocked and worked up with the exclusion of moisture in accordance with Example 1, paragraph Ib. The dried hydrobromide dvat was dissolved in 10 ml of DMF and, after cooling (-10 ° C.), with 155 111 (1.1 mmol Et3N was added and 555 mg (1.5 mmol) of Cbo-Pro-OpNP were added and after a reaction time of 24 hours the reaction solution was evaporated to dryness in vacuo.



  The residue was dissolved in 30 ml of MeOH and filtered through a column of Sephadex LH-20 equilibrated with MeOH. For further purification, the eluate was concentrated in vacuo and the residue was dissolved in 30% AcOH. The solution was filtered through a column of Sephadex G-15. After freeze-drying part of the eluate with the addition of 90, u1 conc. HCI gave 479 mg (61.4% of theory) of an amorphous powder which was uniform in the DSC in the LMS C. Elemental analysis and calculation from the gross formula C34H4lNsO7Cl gave the following values: C = 57.11% (57.58%); H = 5.710 /> (5.83%); N = 16.15% (15.80%); Cl = 4.92% (5.00%).



   The amino acid analysis showed the expected amino acids in the correct ratio: Arg: 0.95 - Phe: 1.00 - Pro: 0.98.



  Example 13 XIII. Na-Bz-Pro-CHA-Arg-pNA HCl CIIIa) N -Cbo-CHA-Arg (NO2) -pNA
9.47 g (20 mmol) of the compound prepared according to Example 1, paragraph Ia were deblocked according to Example 1, paragraph Ib. The dried residue was dissolved in 100 ml of DMF. After cooling, 3.04 g (30 mmol) of Et3N were added to the solution. The HBr-Et3N formed was filtered off. The filtrate was mixed with 9.37 g (22 mmol) of Cbo-CHA-OpNP at -10 "C. The reaction mixture was treated according to Example 1, paragraph Ib. After gel filtration on Sephadex LH-20 in MeOH, 11.72 was obtained g (93.5% of theory) of the crystalline compound XIVa, which melted at 166-168 "C and was uniform in the LMS A and B in the DSC.

  Elemental analysis and calculation from the gross formula C29H38N808 gave the following values: C = 55.20% (55.58%); H = 5.98% (6.11%); N = 18.01% (17.88%).



  XIIIb) Na-Cbo-Pro-CHA-Arg (NO2) -pNA
6.27 g (10 mmol) of compound XIIIa were deblocked according to Example 1, paragraph Ib. The dry hydrobromide derivative was dissolved in 75 ml DMF. After cooling, 2.08 ml (15 mmol) of Et3N was added to the solution. The HBr-Et3N formed was filtered off. 4.07 g (11 mmol) of Cbo-Pro-OpNP were added to the filtrate at -10 ° C. The reaction mixture was further treated according to Example 1, paragraph Ib.



  After gel filtration on Sephadex LH-20 in MeOH, 6.18 g (85.4% of theory) of the amorphous compound XIII was obtained, which was uniform in the LMS A and B in the DSC. Elemental analysis and calculation from the gross formula C34H45N909 gave the following values: C = 56.22% (56.42%); H = 6.14% (6.27%); N = 17.35% (17.42%).



  XIIIc) N -Bz-Pro-CHA-Arg (NO2) pNA
1.45 g (2 mmol) of compound Xlllb were deblocked according to Example 1, paragraph Ib. The dry hydrobromide derivative obtained was dissolved in 15 ml of DMF. After cooling (-10 ° C.), 0.30 g (3 mmol) of Et3N and then 680 mg (3 mmol) of benzoic anhydride were added to the solution. The reaction mixture was further treated according to Example 1, section Id. The crude product obtained was converted into MeOH dissolved and purified by filtration through a column of Sephadex LH-20 in MeOH to give 1.03 g (74.2% of theory) of the amorphous compound XIIIc, which was uniform in DSC in LMS A and B.

  Elemental analysis and calculation from the gross formula G3H43Ns08 gave the following values: C = 57.29% (57.13%); H = 6.30% (6.25%); N = 18.09% (18.17%).



     XIII. Na-Bz-Pro-CHA-Arg-pNA HCl
694 mg (1 mmol) of compound XIIlc were converted to compound XIII according to Example 1, paragraph Ie. Purification: Gel filtration on Sephadex G-15 in 30% AcOH. Yield: 571 mg of freeze-dried amorphous powder (83.3% of theory); uniform in the DSC in the LMS C.



   Elemental analysis and calculation from the gross formula C23H45N8O0Cl gave the following values: C = 57.15% (57.84%); H = 6.49% (6.62%); N = 16.70% (16.35%); Cl = 5.09% (5.17%).



   The amino acid analysis showed the expected amino acids in the correct ratio: Arg: 1.04 - CHA: 1.02 - Pro: 1.00.



  Example 14 XIV. Na-Bz-Pro-Tyr-ArgpNA HCl XIVa) N -Cbo-Tyr (OBzl} Arg (NO2) -pNA
2.37 g (5 mmol) of compound Ia (see Example 1) was deblocked according to Example 1, paragraph Ib. The dried hydrobromide derivative obtained was dissolved in 30 ml of DMF.



  After cooling, the solution was first added to 1.05 ml (7.5 mmol) of Et3N and then 2.82 g (5.5 mmol) of Cbo-Tyr (OBzl> OpNP). The reaction mixture was further treated in accordance with Example 1, paragraph Ib Gel filtration through a column of Sephadex LH-20 in MeOH gave 1.78 g (49.0% of theory) of the amorphous compound XIV), which was uniform in the LMS A and B in the DSC.



   Elemental analysis and calculation from the gross formula C36H3sNsOs gave the following values: C = 59.08% (59.50%); H = 5.11% (5.27%); N = 15.80% (15.42%).

 

  XIVb) Na-Cbo-Pro-Tyr (OBzl} Arg (NO2} pNA
1.25 g (2 mmol) of the compound XlVa were deblocked according to Example 1, paragraph Ib. The hydrobromide derivative obtained was dissolved in 15 ml of DMF. After cooling, 0.30 g (3 mmol) of Et3N and then 815 mg (2.2 mmol) of Cbo-Pro-OpNP were added to the solution. The reaction mixture was treated according to Example 1, paragraph Ib. After gel filtration through a column of Sephadex LH-20 in MeOH, 928 mg (56.3% of theory) of the amorphous compound XIVb were obtained, which was uniform in the DSC in LMS A and B.



   Elemental analysis and calculation from the gross formula c41H45N0Oio gave the following values: C = 56.72% (56.42%); H = 6.09% (6.27%); N = 17.60% (17.42%).



  XIVc) Na-Bz-Pro-Tyr (OBzl} Arg (NO2} pNA
824 mg (1 mmol) of compound XIVb were deblocked according to Example 1, paragraph Ib. The product obtained was dissolved in 10 ml of DMF and 210 μl (1.5 mmol) of Et3N were added.



  After cooling, 340 mg (1.5 mmol) of benzoic anhydride were added to the solution. The reaction product was further treated in accordance with Example 1, paragraph Id. Purification: Gel filtration on Sephadex LH-20 in MeOH. Yield: 422 mg of amorphous substance (53.2% of theory); uniform in the DSC in LMS A and B.



   Elemental analysis and calculation from the gross formula C, 0H43N0O0 gave the following values: C = 60.92% (60.52%); H = 5.38% (5.46%); N = 16.19 / o (15.88%).



  XIV. Na-Bz-Pro-Tyr-Arg-pNA HCl
412 mg (0.5 mmol) of compound XIVc were according to
Example 1, paragraph le, converted to compound XIV Purification: Gel filtration on Sephadex G-15 in 30% AcOH. Yield: 208 mg of freeze-dried amorphous powder (59.8% of theory); uniform in the DSC in the LMS C.



   Elemental analysis and calculation from the gross formula C33H30N8O7Cl gave the following values: C = 57.43% (57.01%); H = 5.78% (5.65%); N = 16.35% (16.12%); Cl = 4.98% (5.10%).



   The amino acid analysis showed the expected amino acids in the correct ratio:
Arg: 0.95 - Tyr: 1.00 - Pro: 0.98.



   Example 15
XV. Na-Bz-Pro-Ph-Gly-Arg-pNA-HCl
XVa) Na-Cbo-PhGly-Arg (NO2) -pNA
2.37 g (5 mmol) of the compound la (see Example 1) were deblocked according to Example 1, paragraph Ib. The hydrobromide derivative obtained was dissolved in 30 ml of DMF. After cooling to -10 C, 1.05 ml (7.5 mmol) of Et3N and then 2.32 g (5.71 mmol) of Cbo-PhGly-OpNP were added to the solution. The
The reaction mixture was treated further in accordance with Example 1, paragraph Ib. After gel filtration through a column of Sepha dex LH-20 in MeOH, 2.62 g (86.4% of theory) of the amorphous compound XVIa were obtained, which were determined in the DSC in the
LMS A and B was uniform.



   Elemental analysis and calculation from the gross formula C28H30NsOs gave the following values: C = 55.10% (55.44%);
H = 5.08% (4.99%); N = 18.81% (18.47%).



   XVb) Na-Cbo-Pro-Ph Gly-Arg (NO2) -pNA
1.22 g (2 mmol) of compound XVa were deblocked according to Example 1, paragraph Ib. The hydrobromide derivative obtained was dissolved in 15 ml of DMF. After cooling, the
300 mg (3 mmol) of Et3N and then 815 mg (2.2 mmol) of Cbo-Pro-OpNP were added to the solution. The reaction mixture was treated according to Example 1, paragraph Ib. After gel filtration through a column of Sephadex LH-20 in MeOH, 1.17 g (83.1% of theory) of the amorphous compound XVb was obtained, which was uniform in the LMS A and B in the DSC.



   Elemental analysis and calculation from the gross formula G3H37NsOs gave the following values: C = 56.08% (56.32%);
H = 5.21% (530%); N = 18.02% (17.91%).



   XVc) Na-Bz-Pro-Ph Gly-Arg (NO2} pNA
704 mg (1 mmol) of compound XVb were deblocked according to Example 1, paragraph Ib. The product obtained was in
10 ml of DMF dissolved and mixed with 210 μl (1.5 mmol) of Et3N.



  After cooling, 340 mg (1.5 mmol) of BzzO were added.



  The reaction product was further treated in accordance with Example 1, paragraph Id. Purification: Gel filtration on Sephadex LH-20 in MeOH. Yield: 518 mg amorphous substance (76.9% of theory); uniform in the DSC in LMS A and B.



   Elemental analysis and calculation from the gross formula C22H35N0O8 gave the following values: C = 56.85% (57.05%); H = 5.19% (5.24%); N = 18.96% (18.71%).



  XV. Na-Bz-Pro-Ph Gly-Arg-pNA HCl
337 mg (0.5 mmol) of compound XVc were converted to compound XV according to Example 1, paragraph Ie. Purification: Gel filtration on Sephadex G-15 in 30% AcOH. Yield: 207 mg of freeze-dried amorphous powder (62.2% of theory); uniform in the DSC in the LMS C.



   Elemental analysis and calculation from the gross formula C32H37NsO6CI gave the following values: C = 57.520 / 0 (57.78%); H = 5.73% (5.6 1%); N = 16.98% (16.85%); Cl = 5.25% (5.33%).



   The amino acid analysis showed the expected amino acids in the correct ratio: Arg: 0.97-Ph-Gly: 1.05-Pro: 1.00.



  Example 16 XVI. Na-Bz-Pro-Phe-Arg-2-NA HCl XVIa) Na-Cbo-Arg (NO2) -2-NA
3.53 g (10 mmol) of well-dried Cbo-Arg (NO2) -OH were dissolved in 150 ml of THF: DMF (3: 1) with exclusion of moisture. After cooling to -10 ° C., 1.39 ml (10 mmol) of Et3N were added to the solution, and then a solution of 1.35 g (10 mmol) of isobutyl chloroformate in 20 ml of THF was added dropwise over the course of 15 minutes, the temperature between - 10 "and -5 ° C. The solution obtained was then added dropwise a solution of 1.72 g (12 mmol) of ss-naphthylamine in 15 ml of THF, while maintaining the above-mentioned temperature. The reaction mixture was prepared according to Example 1, paragraph Ia-2, treated further.

  After gel filtration through a column of Sephadex LH-20 in MeOH, 3.75 g of the crystalline compound XVIa (78.4% of theory) with mp. 173-174.5 C were obtained, which were uniform in the LMS A and B in the DSC was.



   Elemental analysis and calculation from the gross formula C24H26N6O5 gave the following values: C = 60.82% (60.24%); H = 5.63% (5.48%); N = 17.48% (16.72%).



  XVIb) Na-Cbo-Phe-Arg (NO2> 2-NA
2.87 g (6 mmol) of compound XVIa were deblocked according to Example 1, paragraph Ib. The hydrobromide derivative obtained was dissolved in 50 ml of DMF. After cooling to -10 C, 1.25 ml (9 mmol) Et3N was added to the solution. The Et3N-HBr formed was filtered off and washed with a little cold DMF. The DMF solution thus obtained was mixed with 2.78 g (6.6 mmol) of CbO-Phe-OpNP. The reaction mixture was treated according to Example 1, paragraph Ib.



  After gel filtration through a column of Sephadex LH-20 in MeOH, 3.23 g (86% of theory) of the amorphous compound XVlb XVIc) were obtained, which was uniform in the DSC in LMS A and B.

 

   Elemental analysis and calculation from the gross formula C32H35N7O6 gave the following values: C = 63.10% (63.35%); H = 5.54% (5.64%); N = 15.80% (15.67%).



  XVIc. Na-Cbo-Pro-Phe-Arg (N02> 2-NA
1.88 g (3 mmol) of compound XVlb were deblocked according to Example 1, paragraph Ib. The product obtained was dissolved in 20 ml of DMF. The solution was added with 0.63 ml (4.6 mmol) of Et3N and then with 1.22 g (3.3 mmol) of Cbo-Pro-OpNP.



  The reaction mixture was treated according to Example 1, paragraph Ib. After gel filtration through a column of Sephadex LH-20 in MeOH, 1.72 g (79.3% of theory) of the amorphous compound XVIc was obtained, which was uniform in the DSC in LMS A and B.



   Elemental analysis and calculation from the gross formula G8H42N807 gave the following values: C = 62.93% (63.14%); H = 5.82% (5.86%); N = 15.75% (15.50%).



     XVld. Na-Bz-Pro-Phe-Arg (NO2) -2-NA
1.08 g (1.5 mmol) of compound XVIc were deblocked according to Example 1, paragraph Ib. The product obtained was dissolved in 12 ml of DMF and 315 μl (2.25 mmol) of Et3N were added.



  After cooling, 510 mg (2.25 mmol) of Bz20 were added to the solution. The reaction mixture was treated according to Example 1, paragraph Id. Purification: Gel filtration on Sephadex LH-20 in MeOH. Yield: 765 mg amorphous substance (73.6% of theory); uniform in the DSC in LMS A and B.



   Elemental analysis and calculation from the gross formula C37H40Ns06 gave the following values: C = 63.88 / o (64.15%); H = 5.75% (5.82%); N = 16.49% (16.18%).



  XVI. NBz-Pro-Phe-Arg-2-NA-HC1
693 mg (1 mmol) of the compound XVld were converted to the compound XVI according to Example 1, paragraph Ie. Purification: Gel filtration on Sephadex G-15 in 30% AcOH. Yield: 455 mg of freeze-dried amorphous powder (66.5% of theory); uniform in the DSC in the LMS C.



   Elemental analysis and calculation from the gross formula C37Hd2N704C1 gave the following values: C = 65.18% (64.95%); H = 6.13% (6.19%); N = 14.55% (14.330 />); Cl = 5.09% (5.18%).



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



  Example 17 XVII. Na-Bz-Pro-Phe-Arg-4-MeO-2-NA HCl XVIIa) Na-Cbo-Arg (NO2) -4-MeO-2-NA
A solution of 2.08 g (12 mmol) of 4-methoxy-2-naphthylamine in 15 ml of THF was added dropwise to 3.53 g (10 mmol) of Cbo-Arg (NO2) -OH according to Example 16, Section XVIa (instead 2-naphthylamine). After gel filtration through a column of Sephadex LH-20 in MeOH, 3.91 g of the amorphous compound XVIIa (76.9% of theory) were obtained, which was uniform in the DSC in LMS A and B.



   Elemental analysis and calculation from the gross formula C25H2sNOO0 gave the following values: C = 59.20% (59.05%); H = 5.41% (5.55%); N = 16.72% (16.53%).



  XVIIb) Na-Cbo-Phe-Arg (NO2) -4-MeO-2-NA
2.55 g (5 mmol) of compound XVIIa were deblocked according to Example 1, paragraph Ib. The hydrobromide derivative obtained was dissolved in 50 ml of DMF. After cooling to -10 ° C., 1.05 ml (7.5 mmol) of EtN and then 2.31 g (5.5 mmol) of Cbo-Phe-OpNP were added to the solution. The reaction mixture was prepared according to Example 1, paragraph Ib, treated further.



  After gel filtration through a column of Sephadex LH-20 in MeOH, 2.55 g (77.8% of theory) of the amorphous compound XVIIb was obtained, which was uniform in the LMS A and B in the DSC.



   Elemental analysis and calculation from the gross formula C34H37N707 gave the following values: C = 61.95 / o (62.28%); H = 5.62% (5.69%); N = 15.11% (14.95%).



  XVIIc Na-Cbo-Pro-Phe-Arg (NO2) 4-MeO-2-NA
1.97 g (3 mmol) of compound XVIIb) were unblocked in accordance with Example 1, paragraph Ib. The product obtained was dissolved in 20 ml of DMF. The solution was added with 0.63 ml (4.6 mmol) of Et3N and then with 1.22 g (3.3 mmol) of Cbo-Pro-OpNP.



  The reaction mixture was treated according to Example 1, paragraph Ib. After gel filtration through a column of Sephadex LH-20 in MeOH, 1.54 g (68.3% of theory) of the amorphous compound XVIIc was obtained, which was uniform in the DSC in LMS A and B.



   Elemental analysis and calculation from the gross formula C39H44N808 gave the following values: C = 62.05% (62.22%); H = 5.91% (5.89%); N = 15.08% (14.89%).



  XVIId. Na-Bz-Pro-Phe-Arg (NO2) 4-MeO-2-NA
1.13 g (1.5 mmol) of compound XVIIc were unblocked according to Example 1, paragraph Ib. The product obtained was dissolved in 12 ml of DMF and 315 μl (2.25 mmol) of Et3N were added.



  After cooling, 510 mg (2.25 mmol) of BzzO were added to the solution. The reaction mixture was treated according to Example 1, paragraph Id. Purification: Gel filtration on Sephadex LH-20 in MeOH. Yield: 780 mg of amorphous substance (71.9% of theory); uniform in the DSC in LMS A and B.



   Elemental analysis and calculation from the gross formula C3sH42N807 gave the following values: C = 63.48% (63.14%); H = 5.75% (5.87%); N = 15.78% (15.50%).



  XVII. Na-Bz-Pro-Phe-Arg4-MeO-2-NAHCI
723 mg (1 mmol) of the XVIId XVlld. were converted to compound XVII according to Example 1, paragraph Ie. Purification: Gel filtration on Sephadex G-15 in 30% AcOH. Yield: 488 mg of freeze-dried amorphous powder (68.3% of theory); uniform in the DSC in the LMS C.



   Elemental analysis and calculation from the gross formula G8H44N7OsCI gave the following values: C = 63.58% (63.90%); H = 6.15% (6.21%); N = 14.03% (13.73%); Cl = 4.88% (4.96%).



   The amino acid analysis gave the expected amino acids in the correct ratio: Arg: 0.93 - Phe: 1.00 - Pro: 0.94.



   The substrates, e.g. B. the substrate obtained according to Example 1, namely Na-Bz-Pro-Phe-Arg-p NA HCl, was used to determine various enzymes in blood plasma.



  The determination was based on the principle that the cleavage product (NHTR2) 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. Na-Bz Pro-Phe-Arg-p-NA HCI, an absorption maximum at 302 nm (nanometers) and a molar extinction coefficient of 12950. The absorption of the substrate is practically zero at 405 nm. p-Nitroaniline (pNA), 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 extinction coefficient drops only slightly. 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 substrate does not interfere with the measurement at 405 nm. The ratios are practically identical for the other substrates which contain a p-nitroanilino group as the chromophoric group. The spectrophotometric measurement was therefore carried out at 405 nm in all these cases.



   The enzymatic hydrolysis reaction can be shown schematically as follows:
EMI10.1
 E = enzyme S = substrate ES = enzyme-substrate complex P1 and P2 = products kl, k2, k3 and k4 = rate constants dissociation constant for ES = k2 / kl = Km (Michaelis constant) if (S)> (E) and k4 <k3, the following applies:
EMI10.2
 The rate constant at which P1 is formed is: v = kr (ES)
EMI10.3
 If E is completely bound to S, then: (ES) = (E) and v = vmax = k3 <E) (3) Lineweaver-Burk equation:
EMI10.4

From equation (2) it follows that the constants Km and k3 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 you can determine the initial speed of hydrolysis.



   If l / v is plotted as a function of 1 / (S), a Lineweaver-Burk diagram is obtained (see short textbook on biochemistry by P. Karlson, Georg Thieme-Verlag, Stuttgart, 1967, page 70), from which it emerges Have vmax and km determined graphically.



   Km and k3 = vmax / E were determined using Na-Bz Pro-Phe-Arg-p-NA-HCI (substrate according to Example 1) for plasma kallikrein, trypsin, plasmin and thrombin. The results are summarized in Table I.



   Table I
Enzyme activity measured on Na-Bz-Pro-Phe-Arg-pNAHC1 enzyme Km mol / liter vmax umoll ratio
Substrates in mint 5) are common
Units of plasma calli 2.82 10-4 950 10-2 1 E = 4.8 BAEE-E 'pure plasmin 1.4-10 4 32-10-3 I 1E = 518CE4 trypsin 1.7 2.22-10 3 1 E = 12,000 NF2 thrombin 1.6 0.14-10-3 IU = 117600NIH3 1.

  A plasma kallikrein BAEE unit is the amount of enzyme that hydrolyzes one µmol of benzoyl-L-arginine ethyl ester per minute under standard conditions.



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



  3. The thrombin NIH unit is that of the US National Institute of Health.



  4. The plasmin-CE unit is the casein unit that is measured on casein under standard conditions. 5. Enzyme unit 5) Enzyme unit = the amount of enzyme that hydrolyzes 1 µmol of substrate in one minute with substrate saturation.



   In the third column of Table I, the enzyme units determined compared to the substrate according to Example 1 are compared.



   In the accompanying drawings, the figures have the following meanings:
1 is a graphical representation in which the change in the optical density A OD, which is caused by the hydrolytic effect of plasma kallikrein on the substrate according to Example 1 over a period of 30 minutes, is plotted as a function of the amount of plasma kallikrein in a coordinate system.



   FIG. 2 is a graphical representation in which the decrease in the optical density A OD as a function of the amount of protease inhibitor in an aqueous buffered medium with a constant amount of plasma kallikrein and of substrate according to Example 1 is plotted in a coordinate system.



   3 is a graphical representation in which the inhibitory capacity or the induced proteolytic activity of rat plasma after induction of a shock state as a function of time with a constant amount of substrate according to Example 1 is plotted in a coordinate system.



   The dose-response curve in FIG. 1 was determined for different concentrations of plasma kallikrein with a constant concentration of substrate. The measurements were carried out at 25 ° C. and pH 8.2 using a tris-imidazole buffer with an ionic strength of 0.15.



   The measurements, the results of which are recorded in FIG. 2, were carried out as follows: 0.25 ml of plasma callic solution (0.0275 BAEE units / ml) was added to 2.0 ml of tris-imidazole buffer (pH 8.2, Ionic strength = 0.15). Then 0.25 ml protease inhibitor solution (protease inhibitor from lung) was added and the mixture was incubated for exactly 10 minutes at 25 ° C. Then 0.5 ml aqueous substrate solution (0.679 mg of the substrate according to Example 1 per ml) admitted.



  The mixture was incubated at 25 C for 30 minutes. The reaction was then stopped with 1.0 ml of 2N acetic acid and the absorbance was measured using a spectrophotometer at 405 nm.



   The measurements, the results of which are recorded in FIG. 3, were carried out as follows: In order to produce a shock state in a rat, its right hind leg was scalded by immersion in water heated to 80 ° C. for 10 seconds. Blood was then taken from the rat.



  0.25 ml of plasma kallikrein solution (0.022 BAEE units) was added to 2.0 ml of tris-imidazole buffer (pH 8.2, ionic strength = 0.15). Then 0.01 ml of rat plasma was added and the mixture was incubated for 5 minutes at 25 ° C. Then 0.5 ml of aqueous substrate solution (0.679 mg of substrate according to Example 1 per ml) was added and the mixture was incubated at 25 ° C. for exactly 30 minutes . The increase in absorption was measured spectrophotometrically at 405 nm. The measurements were also carried out with a blank sample containing only plasma kallikrein.



   From table list it can be seen that the substrate obtained according to Example 1 has a much higher specificity compared to plasma kallikrein than compared to plasmin, trypsin and thrombin. vmax is much higher for plasma kallikrein than for the other enzymes, which shows that the substrate is hydrolysed much more quickly by plasma kallikrein than by the other enzymes mentioned. This makes it possible to precisely determine small amounts of plasma kallkrein or its inhibitors, which is of great importance in the clinic, where often only small sample amounts are available.



   It can also be seen from the table that thrombin
Determination of plasma kallikrein or its inhibitors does not interfere.



   The substrate also has the advantage that the enzymatic
Hydrolysis by plasma kallikrein the Michaelis-Menten
Law follows and it is possible to determine the kinetic constants Km and vmax.



   1 and 2 show how accurate and sensitive the determination of plasma kallikrein or its inhibitors are. This represents a major advantage in that it becomes possible to continuously monitor the therapy of shock conditions in humans by means of protease inhibitors and to precisely dose their administration.



   FIG. 3 shows how a shock state in which the plasma kallikrein system is activated can be determined by means of the new substrate. The activation of the plasma kallikrein system, which is characteristic of such shock states, can be determined precisely using the new substrate.



   The cleavage product NH2-R2 can first be diazotized before the photometric determination and coupled with a coupling component.



   Table II Plasma kallikrein or human plasmin activity, measured by means of the new substrates at constant substrate concentration and enzyme concentration
BAEE unit
Plasma kallikrein or



     1CE unit human plasmin in
P-Nitroaniline (pNA) formed for 1 minute or



     ss-naphthylamine (2-NA) or



      4-methoxy-B-naphthylamine (4-Meo-2-NA) in nanomoles (nM)
Plasma kallikrein human plasmin le 135.5 nM pNA 152.0 nM pNA II 42.3 nM pNA 49.1 nM pNA III 108.3nMpNA 100.0 nM pNA IV 154.7 nM pNA 158.0 nM pNA V 78 .4 nM pNA 142.0 nM pNA Vl 256.0 nM pNA 144.0 nM pNA VII 59.7 nM pNA 256.0 nM pNA VIII 65.1 nM pNA 600.0 nM pNA IX 88.5 nM pNA 80, 2 nM pNA X 72.1 nM pNA 230.0 nM pNA XI 49.2 nM pNA 196.0 nM pNA XII 109.9 nM pNA 176.1 nM pNA XIII 64.5 nM pNA 116.0 nM pNA X 71, 0 nM pNA 73.8 nM pNA XV 9.1 nM pNA 16.2 nM pNA XVI 44.1 nM 2-NA 95.4 nM 2-NA XVII 39.8 nM 4-MeO-2-NA 79.7 nM 4-MeO-2-NA
The new substrates are expediently not in the amine form,

   but in protonized form, d. H. in the form of a salt with a mineral acid, preferably hydrochloric acid.

 

Claims (1)

PATENT CLAIMS 1. Method for the quantitative determination of proteolytic enzymes of the classification group E.C. 3.4.4. in body fluids of humans and mammals, characterized in that a body is allowed to act on the body fluid, which has the following structure: : R '-Pro-X-Arg-NH-R2 I wherein Rl is an acyl or sulfonyl group, R2 is an aromatic hydrocarbon radical which may have substituents, and X is phenylalanyl, ss-cyclohexylalanyl; Denote phenylglycyl or tyrosyl group, where -NH-R2 represents a chromophoric group, and which has the property, under the hydrolytic action of the enzymes mentioned, a cleavage product of the formula NH2-R2 and that the amount of the product NH2-R2 cleaved by the enzyme is measured by means of photometric, spectrophotometric or fluorescence-photometric methods. 2. The method according to claim 1, characterized in that one uses a substrate whose acyl group denoted by Rl has the following sub-formula: R3 - CO - II, wherein R3 a) an aliphatic hydrocarbon radical with 1 to 17 carbon atoms, b) an araliphatic hydrocarbon radical, the aliphatic part of which contains 1 to 17 carbon atoms and the aryl radical optionally carries an amino group, c) a cycloaliphatic hydrocarbon radical optionally bearing an amino or aminomethyl group, d) an optionally an alkyl, amino or aromatic hydrocarbon radical bearing aminoalkyl group or e) denotes a benzyloxy group. 3. The method according to claim 1, characterized in that one uses a substrate whose sulfonyl group denoted by R 'is a benzenesulfonyl or p-toluenesulfonyl group. 4. The method according to claim 1, characterized in that one uses a substrate in which R2 is a p-nitrophenyl, p-naphthyl or 4-methoxy-p-naphthyl group. 5. The method according to claim 1, characterized in that blood plasma is used as body fluid. 6. A method for determining plasma kallikrein according to claims 1 and 5. 7. The method according to claim 1, characterized in that the cleavage product NH2R2 is diazotized before the photometric measurement and coupled with a coupling component. 8. The method according to claim 1, characterized in that the substrate is used in protonated form. 9. The method according to claims 1 and 8, characterized in that the substrate is used in the form of the hydrochloride. The present invention relates to a method for the quantitative determination of proteolytic enzymes of the classification group E.C. 3.4.4. in body fluids of humans and mammals, in particular for the determination of prekallikrein, prekallikrein activators, kallikrein and kallikrein inhibitors in blood plasma. There are currently various synthetic products as substrates for proteolytic enzymes of the peptide-peptidohydrolase group, in particular those enzymes which cleave the peptide chains on the carboxyl side of arginine and lysine, e.g. B. trypsin, thrombin, plasmin and kallikrein and other class E.C. 3.4.4 (Enzyme gymnastics). E.C. is the abbreviation for Enzyme Committee of the International Union of Biochemistry. According to the type of catalytic reaction that the proteolytic enzymes trigger, i. H. the esterolytic or the amidolytic reaction, the synthetic substrates mentioned can be divided into two main groups, namely the group of the ester substrates and the group of the amide substrates. The ester substrates have hitherto been used far more frequently than the amide substrates for the investigation of reactions in which the enzymes mentioned take part, because the ester substrates are cleaved faster than the amide substrates currently available. Work has been published (see, for example, BWH SEE GERS, Blood Clotting Enzymology, Academic Press, 1967, pp. 36 and 42-44), from which it emerges that the ratio of the reaction rates of the esterolytic and amidolytic catalysis of the enzymes mentioned fluctuates widely is subject. For example, acetylated thrombin has an increased esterolytic activity, while amidolytic activity is almost eliminated. In biological fluids, the esterolytic activity of the enzymes mentioned using low molecular weight ester substrates is not always inhibited to the same extent as the amidolytic activity determined using natural substrates or amide substrates. Using a synthetic chromogenic amide substrate with a structure derived from the structure of the natural substrates, it would be easier to determine the increase or decrease in the enzymatic activity in biological fluids. Natural substrates, such as kininogen and fibrinogen, which usually have high molecular weights, are very difficult to isolate from natural sources in pure native form. The enzymatic hydrolysis of these natural substrates gives rise to products which can be prepared using customary methods, e.g. B. by spectrophotometric measurements, difficult to determine and track. The object underlying the present invention was to develop a synthetic amide substrate which should be easily and rapidly hydrolytically cleavable by plasma kallikrein and which should provide cleavage products which should be easy to determine by photospectrometric methods. To solve this problem, it was assumed that kallikrein in shock states in humans by hydrolytic action from the naturally occurring substrate kininogen (molecular weight about 50,000) releases the biologically highly effective, pain-producing bradykinin (molecular weight about 1000) and that by using a C -terminal structural element of bradykinin and linking this element with a chromogenic or fluorescent group could receive a synthetic amide substrate which has the same or approximately the same susceptibility to plasma kallikrein as the natural substrate and which is chromolytically or amidolysed would form fluorescent fission product with a high molar extinction coefficient measurable at high wavelengths, so that the influence of biological liquids on the measurement would be greatly reduced. ** WARNING ** End of CLMS field could overlap beginning of DESC **.