DK155051B - Tri-peptide derivatives as well as procedure for quantitative analysis of proteolytic enzymes during use thereof - Google Patents

Abstract

Tri-peptide derivatives with the formula: H-D-X-Y-Z-RWhere X is cyclohexylglycyl, cyclohexylalanyl, p-hydroxycyclohexylalanyl, phenylglycyl, phenylalanyl, tyrosyl, leucyl, norleucyl, isoleucyl, valyl, norvalyl, a-aminobutyryl, alanyl, prolyl or pipecolinyl.Y is cyclohexylglycyl, cyclohexylalanyl, p-hydroxycyclohexylalanyl, phenyl glycyl, phenyl alanyl or tyrosyl, and when the meaning of X is limited to cyclohexylglycyl, cyclohexylalanyl, p-hydroxycyclohexylalanyl, phenylglycyl, phenyl alanyl or tyrosyl, as well as leucyl, isoleucyl, norleucyl, valyl, norvalyl, a-aminobutyryl, alanyl, prolyl, or pipecolinyl,Z is arginyl or lysyl, andR is a chromogen group, which can be split in enzymatic hydrolysis, and which can form a coloured or fluorescent compound, is usable as substrates for proteolytic enzyme assay.The tri-peptide derivatives are preferably to be used in the form of their salt with acids.

Description

in

DK 15505 1 B

The present invention relates to novel tripeptide derivatives which are useful as substrates for quantitative analysis for proteolytic enzymes from the enzyme class E.C. 3.4.21, e.g. organ or glandular calluses and plasmin.

5 So-called organ chalice or glandular chalice is produced by various organs and glands, e.g. pancreas, salivary glands, kidneys, intestinal mucosa, etc. and are excreted in the form of a proenzyme or in an active form. These organ or glandular chalice rays differ chemically and physiologically from plasma chalice rays. In certain pathological conditions, the organelle level of secretion is reduced to below or rises above the normal value. Thus, e.g. the uric acid secretion significantly below normal value in patients suffering from kidney disease. In patients suffering from renal cirrhosis, the kallikrein reinsertion is almost completely suppressed. In patients suffering from essential hyper-tension, kallikrein secretion in 24 hours of urine is significantly reduced on average by 50% of normal value (cf., for example, HS Margolius in "Chemistry and Biology of the Kallikrein-Kinin System in Health and Disease" ", 1974, pages 399 - 409). It is therefore important to have simple methods available for a rapid quantitative assay for organ calli-20 cancer. It is known e.g. to carry out assay of urinary alkaline by esteroysis of certain arginine esters, e.g. tosylarginine methyl ester (TAME). In an improved esterolytic process, tosylarginine methyl ester marked with tritium is used and the radioactivity released by the esterolysis is measured. These esterylytic processes have the disadvantage of being unbiological in that the calli-cranes are proteolytic enzymes that cleave natural peptide chains amidolytic but not esterlytic. Furthermore, ester substrates have the disadvantage of being cleaved by several other enzymes, viz. nonspecifically cleaved by kallikrein. The process of using the TAME marker with tritium is complex in that the methanol before the measurement of the radioactivity of the tritium-labeled methanol must be extracted from the esterification mixture with a water immiscible liquid, as the remaining TAME is marked with tritium. which is still left in the esterolysis mixture will otherwise inhibit the measurement.

DK 155051 B i 2 i! .......... In published German Patent Application No. 25,27,932, substrates are described which have the formula R -Pro-X-Arg-NH-R, where R represents a 2 blocking represents a chromogen group and X represents phenylalanyl, β-cyclohexylalanyl, phenylglycyl or tyrosyl.

5 These substrates are very readily cleaved by plasma kallikrein and give a 2. in colored cleavage product R -N1 · ^, the amount of which can be measured by photometric, spectrophotometric or fluorescence photometric methods.

Attempts have also been made to use these substrates as an assay for organ or glandular chalks. However, it has surprisingly proved! in that these substrates are not sensitive to organ or gland kallikrein, ie. not split or only split to a small extent in the latter. ·

Danish patent application no. 3126/76 and Danish patent application no.

2400/73 describes various tripeptide derivatives to be used as substrates by assay for various enzymes. However, the tripeptide derivatives known from Danish patent application 3126/76 are all characterized in that the side chain of the N-terminal amino acid contains a 6-membered aromatic ring. In contrast, the tripeptide derivatives of the present invention have either a hydrogenated 6-membered ring in the N-terminal amino acid or in the middle amino acid, and as shown below, these structural differences have caused an unexpectedly high cleavage rate of the tripeptide derivatives.

Danish Patent Application No. 2400/73 discloses, inter alia, compounds containing phenylalanine as the N-terminal amino acid and valine as the middle amino acid, while the present compounds contain cyclohexylalanine or 4-hydroxycyclohexylalanine as the middle amino acid when the N-terminal amino acid is phenylalanine.

Danish patent application no. 3127/76, cf. DK 146937B relates to tripeptide derivatives which are useful as substrates for the determination of certain proteolytic enzymes, in particular glandular or organ chalcicles and plasmin. The compounds of the invention differ from the compounds known from Danish Patent Application No. 3127/76 in that the N-terminal amino acid or the middle amino acid inevitably carries

DK 155051B

3 is a cyclohexyl, cyclohexylmethyl, 4-hydroxy-cyclohexylmethyl or phenyl radical on the α-carbon atom, while the known compounds either carry valine, proline or Pip radicals in the α-position. Among other things. For example, four compounds may be taken as examples, namely HD-valyl-leucyl-arginyl-5β-nitroanilide dihydrochloride (Compound XVI) (commercially available under the designation S-2266), HD-valyl-leucyl-lysyl-β-nitro-anilide dihydrochloride ( Compound XVIII) (commercially available under the designation S-2251), HD-prolyl-phenylalanyl-arginyl-p-nitroanilide dihydrochloride (Compound XL) (commercially available under the designation 10-S-2302) and HD-pipeclinyl-phenylalanyl-arginyl -p-nitroanilide dihydrochloride (Compound XLI). Compound XVI is a substrate for glandular kallikrein, compound XVIII is a substrate for plasmin, and compounds XL and XLI are substrates for plasmin glandular kallikrein and plasma kallikrein. Comparative tests have shown that the four compounds are enzymatically cleaved at a given amount of glandular kallikrein or plasmin, respectively, at a significantly lower rate than the tripeptide derivatives of the present invention.

The invention accordingly relates to novel chromogenic substrates which have high sensitivity to certain proteolytic enzymes of class E.C. 3.4.21., Especially organ or glandular chalcrins and plasmin, and are therefore useful as substrates for quantitative analysis for these enzymes. Tripeptide derivatives of the invention have the general formula I

H - D - X - Y -Z -R I

Wherein a) X represents alanyl, α-aminobutyryl (But), valyl, norvalyl, leucyl or norleucyl, and Y represents cyclohexylalanyl (CHA), cyclohexyltyrosyl (CHT) or phenylglycyl (Ph'Gly), or b) X represents phenylalanyl or phenylglycyl, and Y represents cyclohexylalanyl or cyclohexylty rosy I,

DK 155051 B

!

J

4 in Z represents arginyl or lysyl, and R represents p-nitrophenylamino, 2-naphthylamino, 4-methoxy-2-naphthylamino, 4-methyl-cumaryl-7-amino, 1,3-di (methoxycarbonyl) phenyl. yl-5-amino, quinolyl-5-amino or 8-nitroquinolyl-5-amino, j5 and its salts with acids, with the proviso that the tripeptide derivative I j cannot be | HD-Val-CHA-Arg-4-methylcumaryl-7-amide, HD-Val-CHA-Arg-1,3-di (methoxycarbonyl) phenyl-5-amide, HD-Val-CHA-Arg-2-naphthylamide, HD-Val-CHA-Arg-4-methoxy-2-naphthylamide, HD-Val-CHA-Arg-p-10 nitroanilide, HD-Val-CHA-Lys-p-nitroanilide or HD-Ph'-Gly-CHA Arg-pNA.

The tripeptide derivatives of formula I are heavily soluble in aqueous media and are therefore preferably used in the form of their salts with acids, especially their salts with mineral acids, e.g. hydrochloric, hydrobromic, sulfuric, phosphoric, etc. or organic acids, e.g. formic acid, acetic acid, propionic acid, trimethylacetic acid, methoxyacetic acid, halogenated acetic acids such as trichloro or trifluoroacetic acid, aminoacetic acid, lactic acid, oxalic acid, malonic acid, citric acid, benzoic acid, core substituted aromatic acids such as toluene acids, The nature of the acid is not critical as the acid does not participate in the reaction between the substrates and the enzymes.

The compounds of formula I and their salts with acids are hydrolytically cleaved by the action of certain proteolytic enzymes of class 3.4.21. (cf. "Enzyme Nomenclature", Elsevier Scientific Publishing Company, Amsterdam 1973, page 238 et seq.), especially organ or glandular calli-crins and plasrin. As a result, a colored or fluorescent cleavage product of the formula R-Nf 2, the amount of which can be measured by photometric, spectrophotometric, fluorescence spectrophotometric or el ect ro nuclei, is produced in various methods. Therefore, the novel compounds are useful for quantitative analysis for proteolytic enzymes from enzyme class E.C. 3.4.21., Which cleaves natural peptide chains on the carboxy side of arginine and lysine, especially organ or glandular chalcrins and 35 plasmin, as well as their inhibitors and proenzymes. 'i

DK 155051 B

5

The invention therefore also relates to a method for quantitative analysis for proteolytic enzymes from enzyme class E.C. 3.4.21., Which cleaves natural peptide chains on the caroboxy side of arginine and lysine, in particular organ or glandular chalice cells and plasmin. The process of the invention is characterized in that the medium is reacted with a tripeptide derivative of the general formula I or a salt thereof and the amount of colored or fluorescent cleavage product R-N1 · formed by the hydrolytic action of the enzyme on the tripeptide derivative is measured by , spectrophotometric, fluorescence spectrophotometric or electrochemical methods. The method according to the invention permits e.g. to analyze for the enzyme content of enzyme preparations or the level of enzymes in human or mammalian body fluids, e.g. urine, pancreas, intestinal mucosa, mammary gland secretion, sweat gland secretion, sputum and blood. By this method, 15 assays can be performed for free organ calli and mucosal calli cleans, as well as callicrins formed from pre-callic clears and further physiological or non-physiological inhibitors of callicrins and physiological or non-physiological activators of callicrins.

The tripeptide derivatives of formula I can be prepared by the methods described below: 1) The chromogenic group R is attached to the carboxy group of C-terminal arginine or lysine, wherein the α-amino group is protected by a protecting group, e.g. a carbobenzoxy or tert-butoxycarbonyl group, the δ-guanidyl group of arginine being protected by proton in-ring, e.g. with HCl, nitration or tosylation, and the ε-amino group for Jysin is protected by a carbenzoxy group, or a p-methyl, p-methoxy or p-chlorobenzyl-oxycarbonyl group or a tert-butoxycarbonyl group. The C-terminal R-NH group also serves as a protecting group during the 30-step synthesis of the peptide chain. The other protecting groups can be selectively removed as necessary to attach additional amino acid derivatives until the desired peptide chain is fully synthesized.

Finally, the remaining protecting groups are completely cleaved without affecting the R-NH group (cf., for example, Miklos Bodansky et al., "Peptide Synthesis", Interscience Publishers, 1966, pp. 163-165).

DK 155051 B

6 in i 2) First, the peptide chain (according to Bodansky et al., Loc. Cit.) Is synthesized, whereby the C-terminal carboxyl group of arginine or lysine is temporarily protected using a conventional ester group, e.g. a methoxy, ethoxy or benzyloxy group for arginine in question or a tert-butoxy group for lysine. j

The ester groups can be removed by alkaline hydrolysis except the tertiary butoxy group, which must be selectively cleaved by trifluoroacetic acid. If the δ-guanidyl group in arginine is protonated, this ester group is enzymatically removed by trypsin, with no racemization going on. Then, the chromogenic R-NH group is attached. PE. When the δ-guanidino group in arginine is protected by a nitro or! tosyl group, or ε-amino group in lysine is protected with a carbobenzoxy or tert-butoxy group, and the N-terminal α-amino group in the tripeptide derivative is protected with a carbobenzoxy group or a 15 p-methyl, p-methoxy or p -chlorobenzyloxycarbonyl group or a tert-butoxy group, all protecting groups are removed at the same time. The removal can be carried out by treating the protected tripeptide derivative with anhydrous HF at room temperature, thus removing all the mentioned protecting groups on the amino and δ-guanidino groups. The removal may also be carried out by treatment with 2N hydrochloric acid in glacial acetic acid at room temperature if the protected tripeptide derivative does not contain nitro or tosyl protecting groups.

The preparation of the tripeptide derivatives of the invention is described in detail in the examples below.

Analyzes of eluates and products prepared according to the Examples are performed by thin-layer chromatography using glass plates coated with silica gel (Merck, F 254). The thin layer chromatograms are developed using the following solvent systems: 30 A Chloroform / methanol (9: 1).

B n-Propanol / ethyl acetate / water (7: 1: 2).

C n -Butanol / acetic acid / water (3: 1: 1).

DK 155051 B

7

The following abbreviations are used:

AcOh = acetic acid

Ala = alanine

Arg = arginine BOC = tert.butoxycarbonyl

But = α-amino butyric acid

Cbo = carbobenzoxy CHA = cyclohexylalanine CHG = cyclohexylglycine CHT = cyclohexyl tyrosine = p-hydroxycyclohexylalanine DMF = dimethylformamide TLC = thin layer chromatography

EtgN = triethylamine HMPTA = phosphoric acid N, N, N ', N', N ", N" -hexamethyltriamide Ile = isoleucine

Leu = leucine SS = solvent system / s

Lys = lysine

MeOH = methanol Nleu = norleucine

Nval = norvalin

OpNP = p-nitrophenoxy

Phe = phenylalanine

Ph'Gly = phenylglycine Pip = pipecolic acid pNA = p-nitroanilide THF = tetrahydro uranium

Tyr = tyrosine

Val = valine 30 Unless otherwise indicated, the amino acids in the peptide chains are L-shaped.

DK 155051 B

8

Example 1.

H-D-CHA-But-Arg-pNA.2HBr.

la. Cbo-Arg-pNA.HCI.

In a 250 ml three-neck flask is dissolved 16.0 g (47.0 millimoles) of Cbo-Arg-5 OH.HCI, which is dried in vacuo over P 2 O, 90 ml of absolute HMPTA

at 20 ° C while keeping the atmosphere in the flask humid. To the resulting solution is added at room temperature first a solution of 4.74 g (47.0 millimoles) of Et 2 N in 10 ml of HMPTA and then portionwise 16.4 g (100 millimoles) of p-nitrophenyl isocyanate (100% excess). After a reaction time of 24 hours at 20 ° C, the majority of HMPTA is removed by distillation in vacuo. The residue is extracted several times with 30% AcOH. The residue is discarded. The combined acetic acid extracts are further purified by passing them through a column of "Sephadex G-15" equilibrated with 30% AcOH and eluted with 30% AcOH. The fraction 15 of the AcOH eluate, which is cleaved by trypsin treatment with release of p-nitroaniline, is lyophilized. Thus, 12.6 g of an amorphous powder is obtained which is homogeneous in solvent system C as shown by TLC.

Calcd for C 20 H 25 N 6 ° 5 Cl: C 51.67 H 5.42 N 18.08 Cl 7.63.

Found: C 51.29 H 5.48 N 17.92 Cl 7.50.

lb. 2HBr.H-Arg-pNA.

4.65 g (10 millimoles) of compound Ia is treated with stirring with 40 ml of 2N HBr in glacial acetic acid for 45 minutes at 20 ° C in the absence of moisture.

The amino acid derivative dissolves during CO 2 evolution. The reaction solution is added dropwise with vigorous stirring to 250 ml of absolute ether. This j results in precipitation of 2HBr. H-Arg-pNA. The etheric phase is suctioned off and the solid phase is washed four times with 100 ml of absolute ether to remove benzyl bromide formed as a by-product and excess HBr and AcOH. The residue is dissolved in 50 ml of methanol, the pH is adjusted to 4.5 by the addition of Et 2 N, and j.

in;

DK 155051 B

The solution is evaporated to dryness in vacuo at 30 ° C. The resulting product is dissolved in 75 mL of MeOH and passed through a column of "Sephadex LH-20" (crosslinked dextrangel) equilibrated with MeOH. From a fraction of the eluate, 4.18 g (91.6% of theory) of amorphous Compound Ib is obtained, which is homogeneous in solvent system C as shown by thin layer chromatography.

Analysis:

Calcd for C12 H20 NgO3 Br2: C 31.60 H 4.42 N 18.43 Br 35.03.

Found: C31.15 H 4.35 N 18.84 Br 34.81.

10 1c. Cbo-CHA-Arg-pNA.HBr.

4.56 g (10 millimoles) of compound Ib are dissolved in 30 ml of freshly distilled DMF and the solution is cooled to -10 ° C. Add 1.40 ml (10 millimoles) of EtgN to the solution with stirring. The Et 2 N.HBr formed is removed by filtration and washed with a small amount of cold dimethylformamide.

4.69 g (11 millimoles) of Cbo-CHA-OpNP are added at -10 ° C to the filtrate, and the reaction is allowed to proceed for 2 - 3 hours in the absence of moisture, whereby the temperature of the reaction solution gradually reaches approx. 20 ° C. The solution is again cooled to -10 ° C, buffered with 0.70 ml (5 millimoles) of EtgN and allowed to react for 2 hours at -10 ° C and for approx. 3 hours at room temperature. This procedure is repeated with 0.70 ml of Et 2 N and after 16 hours the reaction solution is evaporated to dryness in vacuo at 50 ° C. The residue is dissolved in 75 ml of 50% AcOH and purified by gel filtration on a column of "Sephadex G-15" @ equilibrated with 50% AcOH. The major fraction of the AcOH eluate, which is cleaved by treatment with trypsin to release p-nitroaniline, is evaporated to dryness in vacuo at 40 ° C. The residue is dissolved in 150 ml of MeOH and evaporated again to dryness. The resulting residue is dried in a vacuum desiccator at 60 ° C over P2 O5 to give 5.85 g (88.3% of theory) of the amorphous compound 1c, which is homogeneous in solvent system C as shown by TLC.

Analysis:

Calcd for C 29 H 4 () N 706 Br: C 52.57 H 6.09 N 14.80 Br 12.06.

Found: 0 52.28 H 6.16 N 15.09 Br 11.85.

DK 155051 B

10 ld. 2HBr.H-CHA-Arg-pNA. 5.30 g (8 millimoles) of compound 1c is treated with stirring with 32 in j ml 2N HBr in glacial acetic acid for 40 minutes at 20 ° C. The peptide derivative dissolves gradually during (X 3 evolution. The reaction solution is added dropwise 5 with vigorous stirring to 250 ml of absolute ether. This results in precipitation of 2HBr.H-CHA-Arg pNA. The etheric phase is suctioned and the solid phase is washed. four times with 100 ml of absolute ether to remove benzyl bromide formed as a by-product as well as excess HBr and AcOH, the residue is dissolved in 50 ml of MeOH, pH 10 adjusted to 4.5 by Et EtN and the solution evaporate to dryness in vacuo at 30 ° C. The resulting residue is dissolved in 50 ml of MeOH and purified on a column of "Sephadex LH-20" equilibrated with

MeOH. The fraction of the MeOH eluate cleaved by treatment with trypsin under release of p-nitroaniline is evaporated to dryness in vacuo 15 at 30 ° C. The resulting residue is dried in a vacuum desiccator at 40 ° C over P 2 O 5 'to give 4.48 g (91.9% of theory) of amorphous compound Id, which is homogeneous in solvent system C.

Analysis:

Calculated for C C HH HN₂O Br: C, 41.39; H, 5.79; N, 16.09; Br, 26.23.

Found: C 41.80 H 5.86 N 16.31 Br 25.85.

scythe. Cbo-D-CHA-But-Arg-pNA.HBr.

Dissolve 3.05 g (5 millimoles) of compound Id in 20 ml of freshly distilled DMF and cool the solution to -10 ° C. 0.70 ml (5 millimoles) of EtnN is added to the solution with stirring. The EtgN.HBr formed is removed by filtration and washed with a small amount of cold DMF. 1.97 g (5.5 millimoles) of Cbo-D-But.OpNP is added at -10 ° G with stirring to the filtrate. The reaction mixture is allowed to react for 2-3 hours in the absence of moisture, whereby the reaction solution temperature gradually reaches approx. 20 ° C.

The solution is again cooled to -10 ° C, buffered with 0.35 ml (2.5 millimoles) of EtgN and allowed to react for approx. 2 hours at -10 ° C and for another 3 hours at room temperature. This procedure is repeated with 0.35 ml in Et 2 N and after 16 hours the reaction solution is evaporated to dryness in vacuo at 50 ° C. The residue is dissolved in 50 ml of 50% AcOH and purified

DK 155051 B

11 gel filtration on a column of "Sephadex G-15" equilibrated with 50% AcOH. The major fraction of the AcOH eluate, which is cleaved by treatment with trypsin during release, of p-nitroaniline is evaporated to dryness in vacuo at 40 ° C. The residue is dissolved in 100 ml of MeOH and the solution 5 is again evaporated to dryness. The resulting residue is dried in a vacuum desiccator at 60 ° C to give 3.27 g (87.5% of theory) of the amorphous compound 1e, which is homogeneous in solvent system C as shown by TLC.

Calcd for C33H4N807Br: C 53.01 H 6.34 N 14.99 Br 10.69

Found: C 52.88 H 6.40 N 15.28 Br 10.53.

1f. 2HBr.H-D-CHA-But-Arg-pNA.

2.24 g (3 millimoles) of compound le are treated with stirring with 12 ml of 2N HBr in glacial acetic acid for 40 minutes at 20 ° C in the absence of moisture. The tripeptide derivative is gradually dissolved during decarboxylation and CC 2 evolution. The reaction solution is added dropwise with vigorous stirring to 120 ml of absolute ether. This results in the precipitation of 2HBr.H-D-But-CHA-Arg pNA. The etheric phase is aspirated through a filter rod and then the solid phase is washed four times with 50 ml aliquots of absolute ether. The resulting residue is dissolved in 40 ml of MeOH. The pH is adjusted to 4.5 by EtgN and the solution is evaporated to dryness in vacuo at 30 ° C. The residue is dissolved in 30 ml of MeOH and purified on a column of "Sephadex LH-20" equilibrated with MeOH. The fraction of the MeOH eluate cleaved by treatment with 25 trypsin with release of p-nitroaniline is evaporated to dryness in vacuo at 30 ° C. For further purification, the purified residue is dissolved in 30 ml of 33% AcOH and purified by gel filtration on a column of "Sephadex G-15" equilibrated with 33% AcOH. The major fraction of the AcOH eluate, which is cleaved by treatment with trypsin to release p-nitroaniline, is evaporated to dryness in vacuo at 40 ° C. The resulting residue is dried in a vacuum desiccator at 40 ° C above Ρ303 to give 1.71 g (82.1% of theory) of amorphous compound 1f, which is homogeneous in solvent system C as shown by TLC.

i1 12

DK 155051 B

Analysis:

Calcd for C25H47NgO5Br2: C 43.24 H 6.10 N 16.14 Br 23.01

Found: ~ C 43.18 H 6.16 N 16.27 Br 22.73.

i i

The amino acid analysis confirms the presence of the expected amino acids in the correct ratios: Arg: 1.01 - CHA: 0.98 - D-But: 1.00. j i i!

Example 2.

2HBr. H-D-CHA-But-Lys-pNA. i 2a. BOC-Lys (e-Cbo) -pNA.

38.05 g (0.1 mole) of dried oil of BOC-Lys (s-Cbo) -OH is dissolved in a 500 10 ml three-neck flask in 150 ml of absolute HMPTA at 20 ° C in the absence of moisture. To the resulting solution is added at room temperature first a solution of 10.12 g (0.1 mole) of Et 3 N in 25 ml of HMPTA and then portionwise 24.62 g (0.15 mole) of p-nitrophenyl isocyanate (50% excess). ), resulting in a strong CO 2 evolution each time.

After a reaction time of 24 hours at 20 ° C, the majority of HMPTA is removed by distillation in vacuo. The residue is extracted several times with 2% sodium hydrogen carbonate solution and then digested with distilled water. The resulting residue is dried in vacuo at 40 ° C and then extracted several times with hot MeOH until the residue contains only the highly soluble by-product N, N'-bis (p-nitrophenyl) urea. The MeOH extracts are evaporated to 300 ml, whereby some impurities form a flocculent precipitate. After filtration, the filtrate (330 ml) is purified on a column of "Sephadex LH-20" equilibrated with MeOH. The major fraction of the MeOH eluate is evaporated in vacuo at 30 ° C to a small volume, thereby crystallizing a needle-like substance. The obtained crystals are filtered off and washed portionwise with 50 ml of ice-cooled MeOH. After drying in a vacuum desiccator at 40 ° C over P₂O ^ 1.1 g (62.1% of theory) of crystalline compound 2a, melting point ......... which is homogeneous in solvent system A and B as shown by TLC. The mother liquor gives one

DK 155051 B

13 additional amount of 5.8 g (11.6% of theory) of substance 2a, melting point ........ which is homogeneous in solvent systems A and B as shown by TLC.

Analysis: 5 Calculated for ^ 25 ^ 32 ^ 4 ^ 7 * ^ 6.44 N 11.19.

Found: C, 60.23; H, 6.50; N, 11.38.

2b. CFgCOOH.H-Lys (s-Cbo) -pNA.

25.03 g (50 millimoles) of compound 2a is treated with vigorous stirring with 50 ml of freshly distilled anhydrous trifluoroacetic acid at 20 ° C for 60 minutes in the absence of moisture. The BOC group is selectively cleaved during CC 2 development and isobutylene release. The reaction solution is added dropwise with vigorous stirring to 750 ml of absolute ether to precipitate flocculent CF 2 COOH.H-Lys (s-Cbo) pNA. The etheric phase is aspirated through a filter rod. The solid phase is treated four times with 100 ml portions of absolute ether. The resulting residue is dissolved in 200 ml of MeOH. The pH is adjusted to 4.5 by EtgN and the solution is evaporated to dryness in vacuo at

30 ° C. The residue is dissolved in 200 ml of MeOH and purified on a column of "Sephadex LH-20" ® equilibrated with MeOH. The major fraction of the MeOH-20 eluate, which is homogeneous in solvent system C as shown by TLC

evaporated in vacuo at 30 ° C. The resulting residue is dried in a vacuum desiccator at 40 ° C over P2®5 to give 22.64 g (88.0% of theory) of amorphous compound 2b.

Analysis: 25 Calcd for ^ 22 ^ 25 ^ 4 ^ 7 ^ C 51.36 H 4.90 N 10.89.

Found: C, 51.66; H, 4.88; N, 11.08.

2c. BOC-CHA-Lys (ε-Cbo) -pNA.

7.72 (15 millimoles) of compound 2b is dissolved in 50 ml of freshly distilled DMF and cooled to -10 ° C. To the solution is added stirring 6.48 g of 30 (16.5 millimoles) of BOC-CHA-OpNP and 2.09 ml (15 millimoles) of EtgN. The mixture is allowed to react for 3 hours in the absence of humidity, during which the reaction temperature gradually reaches room temperature. The solution is again cooled to -10 ° C and buffered with 1.05 ml (7.5 millimoles) of EtgN. After one ! reaction time of 5 hours repeats this procedure with 1.05 ml Et ~ N. .

ύ I

After a reaction time of 16 hours at 20 ° C, the reaction solution is evaporated to dryness in vacuo at 50 ° C. The residue is dissolved in 150 ml

MeOH and purified by gel filtration on a column "Sephadex LH-20" equilibrated with MeOH. The first major fraction of the MeOH eluate, which is homogeneous in solvent systems A and B as shown by thin layer chromatography, is concentrated to dryness in vacuo at 30 ° C. After drying in a vacuum desiccator at 40 ° C over 30 ° 8.26 g (84.2% of theoretical) amorphous compound 2c which is homogeneous in solvent systems A and B as shown by TLC.

Analysis:

Calculated for C C ^H ^NNN And: C 62.46 H 7.25 N 10.71 Found: C 63.05 H 7.35 N 10.98.

2d. CFgCOOH.H-CHA-Lys (s-Cbo) -pNA.

3.27 g (5 millimoles) of compound 2c are treated with vigorous stirring with 10 ml of freshly distilled anhydrous trifluoroacetic acid for 60 minutes at 20 ° C in the absence of moisture. The reaction solution is added 20 drops with vigorous stirring to 100 ml of absolute ether, thereby amorphous CFgCOOH. H-CHA-Lys (s-Cbo) pNA is precipitated. The etheric phase is sucked off. The solid residue is washed three times with 30 ml portions of absolute ether. The resulting residue is dissolved in 50 ml of MeOH. The pH is adjusted to 4.5 by Et ^N and the solution is evaporated to dryness in vacuo at 30 ° C. The residue is dissolved in 75 ml MeOH and purified on a column of "Sephadex LH-20" equilibrated with MeOH. The major fraction of the MeOH eluate, which is homogeneous in solvent system C as shown by TLC, is evaporated in vacuo at 30 ° C. After drying of the residue in a vacuum desiccator at 40 ° C over P £ 5, 3.06 g (91.6% of theory) of amorphous compound 2d is obtained.

Analysis:

Calcd. For C 31 H 40 N 5 O 8 F 3: C 55.77 H 6.04 N 10.49

Found: C 56.08 H 6.15 N 11.01.

DK 155051 B

15 2nd. Cbo-D-CHA-But-Lys (s-Cbo) -pNA.

2.00 g (3 ml in limol) of compound 2d is dissolved in 15 ml of freshly distilled DMF and cooled to -10 ° C. To the solution is added stirred 1.18 g (3.3 millimoles) of Cbo-D-But-OpNP and 0.42 ml (3 millimoles) of EtnN.

The mixture is allowed to react for 3 hours in the absence of humidity, the temperature gradually reaching 20 ° C. The reaction solution is again cooled to -10 ° C and buffered with 0.21 ml (1.5 millimoles) of Et 2 N. After a reaction time of 5 hours at -10 ° C, the reaction mixture gradually reaches room temperature.

This procedure is repeated with 0.21 ml of Et 2 N, with reaction 10 taking 16 hours. The reaction mixture is evaporated to dryness in vacuo at 50 ° C and the residue is dissolved in 50 ml of MeOH and purified by gel filtration on a column of "Sephadex LH-20" equilibrated with MeOH. The first major fraction of the MeOH eluate, which is homogeneous in solvent systems A and B as shown by TLC, is evaporated to dryness in vacuo at 30 ° C. The residue is dried in a vacuum desiccator at 40 ° C over 2 2 5 'to give 2.12 g (91.4% of theory) of partially crystalline compound 2e.

Analysis:

Calculated for C C ^H ^NgOg C 63.71 H 6.78 N 10.87 Found: C 63.48 H 6.82 N 10.99.

2f. 2HBr.H-But-D-CHA-Lys-pNA.

1.55 g (2 millimoles) of compound 2e is treated with stirring with 12 ml of 2N HBr in glacial acetic acid for 40 minutes at 20 ° C in the absence of moisture. The tripeptide derivative is gradually dissolved with simultaneous cleavage of the two protecting groups Cbo and BOC and (X + evolution. The reaction solution is added dropwise with vigorous stirring to 100 ml of absolute ether, precipitating flocculent 2HBr.HD-But-CHA-Lys pNA. phase is extracted after 30 minutes and the solid phase is washed four times with portions of 25 ml of absolute ether, the resulting residue is dissolved in 40 ml of MeOH, the pH is adjusted to 4.5 by EtgN and the solution is evaporated to dryness in vacuo at 30 DEG C. The residue is dissolved in 30 ml of MeOH and purified on a column of "Sephadex LH-20" equilibrated with MeOH.

DK 155051 B

16 which is cleaved by treatment with trypsin under release p-nitroaniline, evaporated to dryness in vacuo at 30 ° C. For further purification, the purified product is dissolved in 25 ml of 33% AcOH and purified by gel filtration on a column of "Sephadex G-15" equilibrated with 33% 5 AcOH. The fraction of the AcOH eluate which is cleaved by treatment with trypsin to release p-nitroaniline is evaporated to dryness in vacuo at 40 ° C. The resulting residue is dried in a vacuum desiccator at 40 ° C to give 0.99 g (74.3% of theory) of amorphous substance 2f, which is homogeneous in solvent system 10 C as shown by TLC.

Analysis:

Calcd for ^ 25 ^ 42 ^ 6 ^ 5 ^ r2: ^ 45.05 H 6.35 N 12.61 Br 23.98

Found: C 44.75 H 6.39 N 12.67 Br 23.68.

The amino acid analysis confirms the presence of the expected amino acids in the correct ratios: CHA: 0.98 - Light: 1.00 - D-But: 1.02.

A series of other tripeptide derivatives are prepared by the methods described in the above examples. These tripeptide derivatives are grouped in Table I below.

The dipeptide and tripeptide intermediates used to prepare the derivatives shown in Table I are listed in Tables II and III.

17

Table I

DK 155051 B

Forward Analysis

Initial Found Found Calculated% ducts way 5 Eczema (mmol) (ex) Amino acid analysis final product Yield% 3 2AcOH.HD-Val-3e (0.75 mmol) (2f) 84.4 C 54.90 55, 03 CHT-Lys-pNA 2N HBr / AcOH H 7.78 7.70 C30H50N6 ° 10 N 13.05 12.84

Val: CHT: Lys 1.00: 0.98: 1.01 4 2HBr.HD-Leu-4e (1 mmol) (1f) 85.2 C 44.55 44.88 CHA-Arg-pNA 2N HBr / AcOH H 6.53 6.42 C27H46N8 ° 5Br2 N 15.85 15.51

Br, 21.92; 22.12

Leu: CHA: Arg 1.00: 0.98: 0.99 5 2HBr.HD-Niu5e (1 mmol) (1f) 86.0 C 44.75 44.88 CHA-Arg pNA 2N HBr / AcOH H 6.49 6.42 C27H46N8 ° 5Br2 N 15.79 15.51

Br, 21.82; 22.12

Nleu: CHA: Arg 1.00: 0.97: 0.99 25 2HBr.HD-Nval-6e (1 mmol) (1f) 87.1 C 44.39 44.08 CHA-Arg pNA 2N HBr / AcOH H 6.28 6.26 C26H44N8 ° 5Br2 N 16.09 15.82

Br, 22.33, 22.56

NvahCHA: Arg 30 1.00: 0.98: 0.98 18 s 5

DK 15505 1 B

7 2HBr. H-D-Phe-7e (1 mmol) Cif) 82.3 C 47.47 47.63 CHA-Arg-pNA 2N HBr / AcOH H 5.92 5.86 C30H44N8 ° 5Br2 N 15.08 14.81

Br 20.85 21.12 Phe: CHA: Arg 1.00: 0.98: 1.01 8 2HBr.HD-Ala8e (1 mmol) (1f) 86.0 C 41.97 42.36 CHA -Arg-pNA 2N HBr / AcOH H 5.92 5.93 C24H40N8 ° 5Br2 N 16.59 16.47 Br 23.11 23.49

Ala: CHA: Arg 1.00: 0.97: 0.99 9 2HBr. H-D-Nval-9e (1 mmol) (2f) 76.4 C 45.73 45.89 CHA-Lys-pNA 2N HBr / AcOH H 6.61 6.52 C26H44N6 ° 5Br2 N 12.48 12.35

Br 23.18, 23.49

Nval: CHA: Arg 1.00: 0.98: 0.99 (2HBr.HD-Leu10 Cl mmol) (2f) 78.0 C 46.39 46.69 CHA-Lys-pNA 2N HBr / AcOH H 6.63 6.68 C27H46N6 ° 5Br2 N 12.27 12.10

Br, 22.81, 23.01

Leu: CHA: Lys 1.00: 0.97: 1.01 25 11 2HBr.HD-NiuIle (1 mmol) (2f) 77.6 C 46.88 46.69 CHA-Lys pNA 2N HBr / AcOH H 6.72 6.68 C27H46N6 ° 5Br2 N 12.33 12.10

Br, 22.75, 23.01

Nleu: CHA: Lys 30 0.99: 0.97: 1.00 19

Table 11

DK 155051 B

Flow

Initial procedure

Eczema Dipeptide Ducts (Ex.) Analysis 5 µl Precursor (mmol) Yield% Found% Calculated% 3c BOC-CHT-Lys-2b (5 mmol) (2c) 84.3 C 60.48 60.97 (s- Cbo) pNA BOC-CHT-OpNP H 7.11 7.07 C34H47N5 ° 9 (5'5 mmol) N 10.76 10.46 3d CF3COOH.H-CHT-4c (3 mmol) (2d) 90, 8 C 54.08 54.46

Lys (s-Cbo) pNA 6 ml CF3COOH H 6.00 5.90 C31H40N5 ° 9F3 N 10.18 10.14

Table III

20

DK 155051 B

Flow

Initial procedure

Eczema Tripeptide Products (Ex.) Analysis 5 µl Precursor (mmol) Yield% Found% Calculated% 3e Cbo-D-Val-CHT-3d (1.5 mmol) (2e) 77.9 C 62.80 62, 83

Lys (e-Cbo) pNA Cbo-D-Val-OpNP H 6.87 6.78 C42H54N6 ° 10 (1.65 mmol) N 10.61 10.47 10th Cbo-D-Leu-CHA-Id ( 2 mmol) (le) 79.8 C 53.95 54.19

Arg-pNA. HBr Cbo-D-Leu-H 6.70 6.63 C35H51N8 ° 7Br 0pNP (2.2 mmo,) N 14.66 14.45

Br 10.14 10.30 5th Cbo-D-Nleu-CHA-Id (2 mmol) (le) 80.4 C 54.08 54.19 Arg-pNA.Hbr Cbo-D-NIeu-H 6.73 6.63 C35H5lN8 ° 7Br 0pNP (2.2 mmol) N 14.58 14.45

Br 10.08 10.30 6th Cbo-D-Nval-CHA-1d (2 mmol) (le) 82.6 C 53.28 53.61

Arg-pNA. HBr Cbo-D-Nval-H 6.55 6.48 C34H4gNg07Br OpNP (2.2 mmol) N 14.93 14.71

Br 10.25 10.49 7th Cbo-D-Phe-CHA-1d (2 mmol) (le) 84.5 C 56.23 56.36

Arg-pNA.HBr Cbo-D-Phe-H 6.18 6.10 C38H49N8 ° 7Br 0pNP (2.2 mmol) N 14.10 13.84 Br 9.75 9.87 8e Cbo-D-Ala CH1-1d (2 mmol) (le) 88.2 C 52.01 52.39

Arg-pNA.HBr Cbo-D-Ala-H 6.19 6.18 ^ 32 ^ 45 ^ 8 ^ 7Br OpNp (2.2 mmol) N 15.44 15.27

Br, 10.62, 10.89

DK 155051 B

21 9th Cbo-D-Nval-2d (1.5 mmol) (2nd) 90.6 C 63.90 64.10 CHA-Lys (e-Cbo) - Cbo-D-Nval-H 6.98 6.92 pNA OpNP (1.65 mmol) N 10.82 10.68 C42H54N6 ° 9 5 10th Cbo-D-Leu-CHA-2d (1.5 mmol) (2nd) 88.5 C 64.28 64.48

Lys (s-Cbo) pNA Cbo-D-Leu-H 7.11 7.05 C43H56N6 ° 9 OpNP (1.65 mmol) N 10.75 10.49 11th Cbo-D-Nleu-CHA-2d (1 (5 mmol) (2nd) 88.9 C 64.18 64.48

Lys (ε-Cbo) pNA Cbo-D-Nleu-H 7.08 7.05 C43H56N6 ° 9 0pNP (1'65 mmol) N 10.60 10.49

Example 12.

2AcOH. H-D-Nval-CHA-Arg-pNA.

7.09 g (10 millimoles) 2HBr. H-D-Nval-CHA-Arg pNA (prepared according to Example 38) is dissolved in 75 ml of 60% aqueous MeOH. The solution is applied to (i) a column of "Amberlite" JRA-401 in acetate form. The column is eluted with 60% aqueous MeOH, thereby replacing HBr with AcOH by ion exchange.

The eluate is evaporated to dryness in vacuo at 40 ° C. After drying in the vacuum desiccator at 40 ° C over ^ 2 ^ 5 ^, 339 bromide-free 20 2AcOH.H-D-Nval-CHA-Arg pNA (98.5% of theory).

The quantitative enzyme assays using the tripeptide substrates of the invention can be carried out as follows: 1. Urine challic acid assay.

1 ml of urine and 1 ml of TRIS-imidazole buffer with a pH of 7.9 and a 25 ionic strength of 1.0 are incubated at 37 ° C for 5 minutes and then centrifuged to remove sediments.

DK 155051 B

22 1.4 ml of distilled water heated to 37 ° C and 0.4 ml of the centrifugate are well mixed in a plastic cuvette. To this mixture, 0.2 ml of -3 is added to a 2 x 10 M aqueous substrate solution and the ingredients are quickly mixed. This mixture is incubated for exactly 15 minutes at 37 ° C.

The reaction mixture is then mixed with 0.2 ml of glacial acetic acid to stop the enzymatic reaction. For color measurement, a blank is used which consists of the same constituents, but to which is added glacial acetic acid before the addition of the substrate to prevent the enzymatic reaction. The mixed amount of the colored compound R-N1 · 10 is determined photometrically or spectrophotometrically at 405 nm from the difference between the blank and the test sample. Based on the value thus found, urinary alkaline activity in urine is determined using the formula below: AOD., C. x V x 1000 x F 15 min.

15 = mU / ml urine 15 min. x v x ε.

Δ OD = increase in optical density at 405 nm over 15 minutes, V = total volume of the test mixture = 2.2 ml, 20 1000 = conversion factor for conversion of U to mU, F = dilution factor for urine (2), v = volume of sample = 0.4 ml, ε = extinction coefficient divided by 1000 = 10.4.

The calculation of the urinary alkaline content of urine can also be performed by continuously measuring the product R-N1 · (e.g. p-nitroaniline) which is formed. This method is described below for use in assay for glandular potassium in sputum.

In addition to urinary kallic urine, urine also contains urokinase as a proteolytic enzyme that can also cleave the substrate of the invention, albeit to a lesser extent. In the assay method described above, the sum of the activities of urinary kallikrein and urokinase is measured. To obtain an accurate figure for urinary kallicin activity, urokinase activity must be deducted. The latter can be determined in a comparison experiment

DK 155051 B

23 by adding 0.075 units of trypsin inhibitor (bovine lung trypsin inhibitor) per ml. ml of buffer to completely inhibit urinary callic inactivity and measure urokinase activity only.

2. Sputum Gland Calcium Assay: Mix 0.5 ml of sputum with 2 ml of TRIS-imidazole buffer (ionic strength 1.0) and pre-incubate the mixture at 37 ° C for 5 minutes. The incubate is centrifuged. In a test cuvette, place 1.5 ml of distilled water at 37 ° C and add 0.25 ml of the centrifugate. The ingredients mix well.

_3

Then 0.2 ml of a 2 x 10 M aqueous substrate solution is mixed.

10 The change in extinction at 405 nm is then continuously measured for 5 to 10 minutes using a printer. Based on the determined value for AOD per per minute the callikin activity is calculated per minute. ml of sputum in mU using the following equation: AOD. x V x 1000 x F min.

15 = mU / ml sputum v x ε.

F = 5 V = 1.95 v = 0.25 20 1 U (unit) = amount of enzyme capable of cleaving 1ymol of substrate in 1 minute under optimal or otherwise defined pH conditions, ionic strength, temperature and substrate concentration.

In pancreas, pancreatic kallikrein is present mainly in the form of precallikrein, for which assay can only be performed after activation, e.g. using trypsin. Following activation of precallikrein, trypsin is inhibited by soybean trypsin inhibitor (SBTI). The potassium content of this activated mixture can be determined by one of the methods described above.

3. Plasmin Assay: Mix 1.7 ml of TRIS-imidazole buffer (pH 7.5, ionic strength 0.2) with 0.1 ml of a solution of plasmin in 25% glycerol at 37 ° C, and bian-! 24

DK 155051 B

The things are incubated at 37 ° C for 1 minute. 0.2 ml of an aqueous 2 x 10 M substrate solution at 37 ° C is added to the mixture and the ingredients are quickly mixed. The amount of cleavage product R-N1 · 4 released from the substrate per unit of time is then continuously measured. From the 5 value determined per Plasma activity per minute is calculated per minute. ml sample in mU based on the following equation: ΔΕ / min. x V x 1000 - = mU / ml sample v x ε.

10 ΔΕ = amount of cleavage product released per minute, V = total volume of test mix, v = volume of sample, ε = extinction coefficient divided by 1000.

4. Human plasma antiplasmin assay: 0.1 ml of plasma diluted with 1:20 TRIS-imidazole buffer is mixed with 0.02 ml of a solution of 1.25 CU of human plasmin (preparation from AB Kabi, Stockholm, Sweden) and 50 ATU hirudin (preparation from Pentapharm AG, Basel, Switzerland) per ml in 25% glycerol. The mixture is incubated for 90 seconds at 37 ° C. The incubate is mixed with 1.7 ml of TRIS-imidazole buffer (pH 7.5, ionic strength 0.2) at 37 ° C and then with 0.2 ml of a 2 x 10 µM aqueous substrate solution. The amount of cleavage product R-N1 · 4 released from the substrate per time unit is then continuously measured. From the determined value, the residual plasmin activity is calculated in the manner described above.

In a blank test, the plasma is replaced by the corresponding amount of buffer, but otherwise the test is performed in the manner described above. The particular plasmin activity corresponds to the starting plasmin activity. The anti-plasmin activity is calculated from the difference between the plasmin activity 30 determined in the blank and the remaining plasmin activity determined in the test using the plasma of the following formula:. 25

DK 15505TB

(4Eblind sample - 4Eplasm sample) / min · x V x F x 1000 = mlU / ml v x ε plasma F = plasma dilution factor (20).

The substrates of the invention can also be used to assay for plasminogen in human plasma by converting plasminogen present in the plasma by urokinase or streptokinase into a buffer system and determining the amount of plasmin formed by one of the substrates of the invention according to the plasmin assay method described above. The amount of plasminogen initially present in the plasma is derived from the value determined for plasmin, as after activation one molecular plasmin is formed from one molecular plasminogen.

In Table IV below, the sensitivity of some of the substrates 15 according to the invention to organ or glandular chalice and plasmin is indicated.

Table IV

Urinary kallikrein activity in 1 ml of human urine 0, submandibular kallikrein in 1 ml of sputum, human plasmin and human NIH thrombin, as measured by the substrates of the invention at constant substrate and enzyme concentrations. In comparison, the corresponding values for the known substrates are 2HCI.HD-Val-Leu-Arg-pNA (A), 2HCI.HD-Val-Leu-Lys-pNA (B), HD-Pro-Phe-Arg-pNA ( C) and HD-Pip-Phe-Arg-pNA (D) (cf. Danish Patent Application No. 3127/76) are also indicated.

-4 25 Substrate Concentration: 2 x 10 M.

DK 155051 B

26 Quantity in nanomoles of the R-N1 · 1 releasing product released in 1 minute of 1 ml of human urine, 1 ml of human sputum and 1 CU unit of human plasmin, respectively.

5 Urinary Submandibula- Human kallikrein rice kallikrein plasmin A 0.90 12.2 83 B 0.30 5.7 347 10 C 0.81 - 451 D 1.08 - 401

Substrates of Example 1 2.99 31.2 623 2 2.22 21.0 532 3 1.40 16.5 532 20 4 3.11 35.9 1069 5 2.08 28.1 1179 6 3.12 41 , 3 969 7 2.14 28.5 814 8 1.73 22.4 433 25 9 1.87 18.04 655 10 1.73 19.3 778 11 0.77 8.4 695 ° Urine sample obtained by mixing 100 ml portions of morning urine from healthy people.

The measurement of the cleavage product R-N 2 formed by the enzymatic hydrolysis of the substrate is based on the assumption that

Claims (6)

With substrates containing a 2-naphthylamino, 4-methoxy-2-naphthylamino, 4-methyl-cumaryl- (7) -amino or 1,3-di (methoxycarbonyl) phenyl- (5) -amino group the amount of the cleavage product R-NH2 is measured by fluorescence spectrophotometry. In a test system consisting of enzyme, buffer and substrate, the emitted light with lower energy 15 is measured at 400-470 nm after the resulting fluorescent cleavage product is excited with higher energy light. The amount of cleavage product formed per time unit is measured for the existing enzyme activity. As defined, 1 µmol of cleavage product corresponds to per minute to 1 enzyme unit, calculated on a given substrate. 20 PATENT REQUIREMENTS A tripeptide derivative of the general formula IH - D - X - V -Z-R 1 where a) X represents alanyl, α-aminobutyryl (But), valyl, norvalyl, leucyl or norleucyl, and Y represents cyclohexylalanyl (CHA), cyclohexyl tyrosyl (CHT) or phenylglycyl (Ph'Gly), or b) X represents phenylalanyl or phenylglycyl and Y represents cyclohexylalanyl or cyclohexyl tyrosyl. DK 155051 BZ represents arginyl or lysyl and R represents p-nitrophenylamino, 2-naphthyl methoxy-2-naphthylamino, 4-methyl-cumaryl-7-amino, 1,3-di (methoxycarbonyl) phenyl-5-amino, quinolyl-5-amino or 8-nitroquinolyl-5-amino, 5 and salts thereof with acids, with the proviso that the tripeptide derivative I cannot be HD-Val-CHA-Arg-4-methylcumaryl-7-amide, HD-Val-CHA-Arg-1,3-di (methoxycarbonyl) phenyl 5-amide, HD-Val-CHA-Arg-2-naphthylamide, HD-Val-CHA-Arg-4-methoxy-2-naphthylamide, HD-Val-CHA-Arg-p-10 nitroanilide, HD-Val-CHA -Lys-p-nitroanilide or HD-Ph'Gly-CHA-Arg-p-nitroanilide. Tripeptide derivatives according to claim 1, characterized in that the dipeptide fragment attached to Arg or Lys is Ala-CHA, Ala-Ph'Gly, But-CHA, Val-CHA, Val-CHT, Nval-CHA, Nval-Ph'Gly, Leu-CHA, Leu-Ph'Gly, Nleu-CHA, Phe-CHA, Ph'Gly-CHA or Ph'Gly, -CHT. Tripeptide derivatives according to claim 1 or 2, characterized in that they are protonated with a mineral acid, e.g. hydrochloric acid, hydrobromic acid, sulfuric or phosphoric acid, or an organic acid, e.g. formic acid, acetic acid, propionic acid, trimethylacetic acid, methoxyacetic acid, halogenated acetic acids such as trichloro or trifluoroacetic acids, aminoacetic acid, lactic acid, oxalic acid, malonic acid, citric acid, benzoic acid, in the nucleus substituted aromatic acids such as toluene acids, . 4. Process for Quantitative Analysis for Proteolytic Enzymes from Enzyme Class E.C. 3.4.21., Which cleaves natural peptide chains on the carboxyl side of arginine and lysine, in media containing the enzymes, characterized in that the medium is reacted with a tripeptide derivative of formula I or a salt thereof according to claim 1 or 2, and The amount of colored or fluorescent cleavage product R-N1 · formed by the hydrolytic action of the enzyme on the tripeptide derivative is measured by photometric, spectrophotometric, fluorescence spectrophotometric or electrochemical methods. A method according to claim 4, characterized in that it is analyzed for organ or chiral chalciferous cells in human body fluids, e.g. urine, pancreas, intestinal mucosa, mammary gland secretion, sweat gland secretion, sputum or blood. A method according to claim 4, characterized in that the plasma or blood plasma is analyzed.