NO155540B - NEW TRIPEPTID DERIVATIVES, AND USE THEREOF FOR QUANTITATIVE DETECTION OF PROTEOLYTIC ENZYMES. - Google Patents

Description

The present invention relates to new tripeptide derivatives which

are applicable as substrates for the quantitative determination of proteolytic enzymes, especially enzymes from the class E.C. 3.4.21., e.g. organ or gland kallikrein, thrombin and plasmin.

So-called organ kallikrein or glandular kallikrein is produced

of various organs and glands, e.g. the pancreas, salivary gland, kidney, the mucus in the digestive tract, etc., and is secreted in the form of a proenzyme or in an active form. These organ or glandular kallikreins differ chemically and physiologically from plasma kallikrein. In certain pathological conditions, the level of organ kallikrein secretion falls below or rises above the normal value. Thus falls, e.g. kallikrein secretion in urine significantly below the normal value in patients suffering from kidney diseases. In patients suffering from cirrhosis of the kidney, kallikrein secretion has almost completely ceased. In patients suffering from significant hypertension, kallikrein secretion in 24-hour urine is significantly reduced, usually by approx. 30% average of the normal value (cf. e.g. H.S. Margolius in "Chemistry and Biology of the Kallikrein-Kinin System in Health and Disease", 1974, pages 399-409. Therefore, it is important to have available simple methods for rapid quantitative determination of organ kallikrein. It is known, for example, to measure urinary cabbage kallikrein by esterolysis of certain arginine esters, e.g. tosyl-arginine methyl ester (TAME). In an improved esterolytic method, tosyl-arginine methyl ester labeled with tritium and the radioactivity of the methanol released by esterolysis is measured. These esterolytic methods suffer from the disadvantage that they are not biological insofar as the kallikreins are proteolytic enzymes that cleave natural peptide chains amidolytically but not esterolytically. Furthermore, the ester substrates have the disadvantage that they are cleaved by many other enzymes, i.e. ., is non-specifically cleaved by kallikrein. The method using TAME labeled with tritium is complex as far as methanol before the measurement of the radioactivity in the methanol which e r labeled with tritium must be extracted from the esterolysis mixture with a liquid immiscible with water because the remainder of TAME labeled with tritium

which is still present in the esterolysis mixture would otherwise inhibit the measurement.

In DOS No. 25 27 932, substrates with the formula R<1->Pro-X-Arg-2 1 2 NH-R are described in which R means a blocking group, -NH-R means a chromogenic group and X means a phenylalanyl, 3-cyclohexylalanyl, phenylglycyl or tyrosyl group. These substrates are very easily cleaved by plasma kallikrein and give a colored cleavage product R 2 -NH2 whose quantity can be measured by photometric, spectrophotometric or fluorescence photometric methods. An attempt was also made at

to use these substrates for the determination of organ or glandular kallikreins. However, it was surprisingly found that these substrates are not sensitive to organ or glandular kallikrein, i.e. not cleaved or only to a small extent by the latter.

In DOS No. 26 29 067, tripeptide derivatives are described which are usable as substrates for the determination of certain proteolytic enzymes, e.g. glandular or organ kallikreins and plasmin. Two examples are mentioned there, namely H-D-valyl-leucyl-arginyl-p-nitroanilide dihydrochloride and H-O-valyl-leucyl-lysyl-p-nitroanilide dihydrochloride. The first tripeptide derivative is a substrate for glandular kallikrein, while the second tripeptide derivative is a substrate for plasmin. The two compounds are split by these enzymes and give p-nitroaniline, the amount of which can be measured photometrically or spectrophotometrically. However, the sensitivity for H-D-valyl-leucyl-arginyl-p-nitroanilide dihydrochloride only just reaches the threshold required for accurate determination of urinary kallikrein in unconcentrated urine. However, if this amide substrate is used in concentrated urine, the photometric measurement of p-nitroaniline is greatly interfered with by the intrinsic color of the urine.

The first two amino acids in the tripeptide chain of the two tripeptide derivatives mentioned in the above-mentioned DOS 26 29 067 have hydrophobic isopropyl groups in the a- or [3-position. Attempts were made to replace the isopropyl groups with aromatic groups, e.g. phenyl groups, while maintaining the basic structure of the dipeptide chain to increase hydrophobicity and, consequently, sensitivity to glandular kallikrein. However, this attempt failed. The tripeptide substrates obtained by introducing aromatic groups are not cleaved at all or are cleaved to a very small extent by glandular kallikrein. However, these tripeptide substrates with aromatic groups have a surprisingly high sensitivity to plasmin. Furthermore, it was surprisingly found that the hydrogenation of the aromatic residues in these tripeptide plasmin substrates leads to new tripeptide substrates which have a surprisingly high sensitivity to organ or glandular kallikreins. It had been assumed that in the construction of tripeptide chains only naturally occurring amino acids could be used to provide substrates that would be cleaved by proteolytic enzymes. Therefore, it was surprising to find that by using amino acids that contain cyclohexyl residues and which do not occur in nature, one could obtain chromogenic substrates that are easily cleaved by organ or glandular kallikreins and other proteolytic enzymes, e.g. plasma kallikrein.

The present invention relates to new chromogenic substrates which have a high sensitivity to certain proteolytic enzymes, especially enzymes from the enzyme class E.C. 3.4.21., e.g. organ or gland kallikreins, plasmin and thrombin and are therefore applicable as substrates for the quantitative determination of these enzymes. These substrates are tripeptide derivatives with the formula:

where

a) X represents a cyclohexylglycyl, cyclohexyl-alanyl or cyclohexyltyrosyl group, and

Y represents an alanyl, α-aminobutyryl, norvalyl,

leucyl, norleucyl or pipecoline group or

b) x represents a cyclohexylglycyl, cyclohexylalanyl, cyclohexyltyrosyl or phenylglycyl group,

and

Y represents a phenylalanyl or phenylglycyl group, or

c) X represents an alanyl, α-aminobutyryl, valyl, norvalyl, leucyl or norleucyl group, and

Y represents a cyclohexylalanyl, cyclohexyltyrosyl or phenylglycyl group, or

d) X represents a phenylalanyl or phenylglycyl group, and

Y represents a cyclohexylalanyl or cyclohexyltyrosyl group,

Z represents an arginyl or lysyl group and

R represents p-nitrophenylamino, 2-naphthylamino, 4-methoxy-2-naphthylamino, 4-methyl-coumaryl-7-amino, 1,3-di(methoxycarbonyl)phenyl-5-amino, quinonyl-5-amino or 8-nitroquinonyl-5-amino and salts thereof with acids, with the proviso that the tripeptide derivative cannot be

H-D-CHG-Phe-Lys-pNA, H-D-Va1-CHA-Arg-MCA,

H-D-CHG-Phe-Lys-MCA, H-D-Val-CHA-Arg-DPA,

H-D-CHG-Phe-Lys-DPA, H-D-Val-CHA-Arg-2-NA,

H-D-CHG-Phe-Lys-2-NA, H-D-Val-CHA-Arg-4-MeO-2-NA, H-D-CHG-Phe-Lys-4-MeO-2-NA, H-D-CHG-Leu- Arg-pNA, H-D-Val-CHA-Arg-pNA, H-D-Val-CHA-JLys-pNA,

H-D-CHG-Phe-Arg-pNA, H-D-CHT-Phe-Arg-pNA,

H-D-Ph'-Gly-CHA-Arg-pNA, H-D-CHG-Leu-Lys-pNA or H-D-CHT-Pip-Arg-pNA.

The tripeptide derivatives of formula I are difficult to dissolve in aqueous media and are therefore preferably used in the form of salts with acids, especially the salts with mineral acids, e.g. HC1, HBr, H2SO4, H3PO4 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, aromatic acids substituted in the nucleus such as toluene acids, chloro- or bromobenzoic acids, methoxybenzoic acids and aminobenzoic acids, phthalic acid etc. The nature of this acid is not critical because the acid does not participate in the reaction between the substrates and the enzymes.

The substrates of formula I and their salts with acids are hydrolytically cleaved under the action of certain proteolytic enzymes, especially enzymes of the class 3.4.21. (cf. "Enzyme Nomenclature", Elsevier Scientific Publishing Company, Amsterdam 1973, page 238 ff), e.g. organ and glandular kallikreins, plasmin and thrombin, and as a result a colored or fluorescent cleavage product of the formula R-NH2 is formed whose quantity can be measured by photometric, spectrophotometric, fluorescent spectrophotometric or electrochemical methods. Therefore, the new substrates are applicable for the quantitative determination of proteolytic enzymes, especially enzymes from the class E.C. 3.4.21., which cleaves natural peptide chains on the carboxyl side of arginine as well as lysine, e.g. organ or gland kallikreins, plasmin, plasma kallikrein, thrombin as well as their inhibitors and proenzymes, and also other factors that participate in the formation or inhibition of these enzymes.

The tripeptide derivatives described in the following examples 1, 2, 4, 6, 7, 8, 9, 10, 23, 29 and 30 can be used in particular as substrates for the determination of urinary potassium

crazy.

For the determination of kallikrein in human sputum, the tripeptide derivatives described in the following embodiment examples 1, 2, 4, 6, 7, 8, 9, 10, 15, 16, 17, 18, 19, 20, 23, 25, 26 , 27, 28, 29, 30, 32, 33 and 34 are used.

The tripeptide derivatives described in the following examples 1, 3, 5, 6, 7, 9, 13, 14, 21, 22, 25, 26, 29, 30 and 31 form a group of substrates that can be used to determine plasmin .

The tripeptide derivatives described in the following examples 11, 12, 13, 14, 18, 19, 20, 23, 24, 26, 27, 28, 32, 33 and 34 are very sensitive substrates for the determination of thrombin.

The invention also relates to the use of the tripeptide derivative for the quantitative determination of proteolytic enzymes from the enzyme class E.C. 3.4.21., which cleaves natural peptide chains on the carboxyl side of arginine as well as lysine, e.g. organ or gland kallikreins, plasmin and thrombin. The method of application consists in reacting the material containing the above-mentioned enzymes or in which the latter is formed or consumed with a tripeptide derivative of formula I and measuring the amount of colored or fluorescent cleavage product R-NH2 which is formed by the hydrolytic action of the enzyme on the tripeptide derivative by photometric, spec -troph otometric, fluorescence spectrophotometric or electrochemical methods. The procedure possible-

makes a determination of e.g. the enzyme content in enzyme preparations or the amount of enzyme in the body fluids of humans and mammals, e.g. urine, pancreatic saliva, digestive mucous membranes, mammary gland secretion, sweat gland secretion, saliva and blood. The method is particularly applicable for quantitative

tative determination of organ or glandular kallikreins in the above-mentioned body fluids and plasma kallikrein. With help

of this method, free organ kallikreins and mucosal kallikreins as well as kallikreins that are formed from pre-kallikreins and further physiological or non-physiological inhibitors for kal-

likreins and physiological or non-physiological activators of prekallikreins are determined.

The tripeptide derivatives of formula I can be prepared according to the methods described below: (1) The chromogenic group R is attached to the carboxyl group of C-terminal arginine or lysine, whereby the α-amino group is protected by a protecting group, e.g. a carbobenzoxy or tert.-butoxycarbonyl group, the 6-guanidyl group in the case of arginine is protected by protonation, e.g. with HCl, nitration or tosylation, and the £-amino group in the case of lysine is protected with a carbobenzoxy group, or a p-methyl, p-methoxy or p-chlorobenzyloxycarbonyl group, or a tert-butoxycarbonyl group. The C-terminal R-NH group also serves as a protecting group during the stepwise synthesis of the peptide chain. The other protecting groups can be selectively removed as needed to attach the further amino acid derivatives until the desired peptide chain is completely synthesized. Optionally, the remaining protecting groups are completely cleaved off without affecting the R-NH group (cf. e.g. Miklos Bodansky et al., "Peptide Synthesis", Inter-science Publishers, 1966, pages 163-165).. (2) . First, the peptide chain is synthesized (according to Bodansky et al., loe.eit.) during which, however, the c-terminal carboxyl group in arginine or lysine is protected with a common ester group, e.g. a methoxy, ethoxy or benzyloxy group in the case of arginine, or a tert-butoxy group in the case of lysine. The ester groups can be removed by alkaline hydrolysis, except for the tert.-butoxy group, which is selectively removed,

using trifluoroacetic acid. If the &-guanidyl group in arginine is protonated, this ester group is removed enzymatically by trypsin, and no racemerization takes place. The chromogenic group R-NH- is then attached. If the &-guanidino group in arginine is protected with a nitro or tosyl group, or the £-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 carboben-

oxy group or a p-methyl, p-methoxy or p-chlorobenzyloxycarbonyl group or a tert.-butoxy group, all protecting groups are removed simultaneously. The removal can be carried out by treating the protected tripeptide derivative with anhydrous HF at room temperature, and all the above-mentioned protecting groups for amino and & -guanidino groups are thus removed. The removal can also be carried out by treatment with 2N HBr in glacial acetic acid at room temperature if the protected tripeptide derivative contains no nitro or tosyl protecting group.

The preparation of the tripeptide derivatives according to the invention is described in detail in the following examples.

The analysis of the eluates and products obtained according to the examples was carried out by thin-layer chromatography using a glass plate coated with silica gel (Merck, F 2 541. The thin-layer chromatograms were developed with the following solvent systems:

A chloroform/methanol (9:1)

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

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

The following abbreviations are used:

AcOH = acetic acid

Ala = alanine

Arg = arginine

BOC = tert-butoxycarbonyl

But = α-aminobutyric acid

Cbo = carbobenzoxy

CHA = cyclohexylalanine

CHG = cyclohexylglycine

CHT = cyclohexyltyrosine = p-hydroxycyclohexylalanine DMF = dimethylformamide

DPA = 1,3-di(methoxycarbonyl)-phenyl-(51-amide).

TLC = thin layer chromatography

Et^N = triethylamine

HCA = p-hydroxycyclohexylalanine HMPTA = phosphoric acid N,N,N',N',N",N"-hexamethyltriamide Ile = isoleuciri

Leu = léucite

SS = soluble system(s)

Lys = lysine MCA = 4-methyl-coumaryl-(7)-amide MeOH ' = methanol

4-MeO-2-NA= 4-methoxy-2-naphthylamide Nleu = norleucine

Nval = norvaline

OpNP p-nitrophenoxy

Phe = phenylalanine

Ph'Gly = phenylglycine

Pip = pipecolic acid pNA = p-nitroanilide THF = tetrahydrofuran

Tyr = tyrosine

Val = valine

Unless otherwise stated, the amino acids in the peptide chains have the L-form.

Example 1

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

let. Cfao-Arg-pNA.HC1

r a 250 ml three-necked flask was 16.0 g (47.0 mmol) Cbo-Arg-OH. HC1, which had been dried in vacuo over P2°5'°PP*<*st * 90 ml of absolute HMPTA at 20°C while the atmosphere in the flask was kept free of moisture. The resulting solution was first added at room temperature to a solution of 4.74 g (47.0 mmol) of Et^N in 10 ml of HMPTA and then 16.4 g (100 mmol) of p-nitro-phenyl isocyanate (100% excess) in portions. After a reaction time of 24 hours at 20°C, the main part of HMPTA was removed by distillation in vacuum. The residue was extracted several times with 30% AcOH. The rest was thrown away. The combined acidic extracts were further purified by passage through a column of "Sephadex G-15" (Trademark) equilibrated with 30% AcOH and eluted with 30% AcOH. The fraction of the AcOH eluate which was cleaved by treatment with trypsin to release p-nitroaniline was freeze-dried. This resulted in 12.6 g of an amorphous powder which is homogeneous in SS C as shown by TLC. Elemental analysis and calculation from the empirical formula C20<H>25<N6>°5C"''^a the ^^' 3en^ B values (the values from the empirical formula are set in parentheses) : C = 51.29% (51.67%), H = 5.48% (5.42%),, N - 17.92% (18.08%) , Cl = 7.50% (7.63%)_.

Ib. 2HBr. H-Arg-pNA

4.65 g (10 mmol) of compound 1a was treated with stirring with 40 ml of 2NHBr in glacial acetic acid for 45 min. at 20°C in the absence of moisture.. The amino acid derivative resolved under CO., evolution. The reaction solution was added dropwise with vigorous stirring to 250 ml of absolute ether. This resulted in precipitation of 2HBr. H-Arg-pNA. The ether phase was suctioned off, after which the solid phase was washed 4 times with 100 ml portions of absolute ether to remove benzyl bromide which had formed as a by-product together with excess HBr and AcOH. The residue was dissolved in 30 ml of methanol, pH adjusted to 4.5 by addition of Et₂N, and the solution was concentrated to dryness in vacuo at 30°C. The resulting product was dissolved in 75 ml of MeOH and passed through a column of "Sephadex LH-20" (cross-linked dextran) equilibrated with MeOH. From a fraction of the eluate one obtained

4.18 g (91.6% of theory) of amorphous compound 1b which was homogeneous in SS C as shown by TLC. Elemental analysis and calculation from the empirical formula ci2H2 0N6°3Br2 ^a ^e f0!9nding values: C = 31.15% (31.60%), H = 4.35% (4.42%1, N = 18, 84% (18.43%) and Br = 34.81% (35.03%).

lc. Cbo-CHA-Arg-pNA. HBr

4.56 g (10 mmol). of compound 1b was dissolved in 30 ml of freshly distilled DMF and the solution was cooled to -10°C. 1.40 mL (10 mmol) of Et₂N was added to the solution with stirring. The formed Et.jN.HBr was removed by filtration and washed with a small amount of cold DMF. 4.6 9 g (11 mmol) Cbo-CHA-OpNP was added at -10°C to the filtrate, and the reaction was allowed to continue for 2 to 3 hours without moisture, during which the temperature in the reaction solution gradually reached approx. 20°C. The solution was again cooled to -10°C, buffered with 0.70 ml (5 mmol)Et3N and allowed to react for approx. 2 hours at -10°C and approx. 3 hours at room temperature. This procedure was repeated with 0.70 ml Et₂N, and after 16 hours the reaction solution was concentrated to dryness in vacuo at 50°C. The residue was dissolved in 75 ml of 50% AcOH and purified by gel filtration on a column of "Sephadex G-15" equilibrated with 50% AcOH. The main fraction of the AcOH eluate which was cleaved by treatment with trypsin to release p-nitroaniline was concentrated to dryness in vacuo at 40°C. The residue was dissolved in 150 ml of MeOH and again concentrated to dryness. The resulting residue was dried in a vacuum desiccator at 60°C over P2°5og9a 5'85 g (88.3% of theoretical yield), amorphous compound 1c which was homogeneous in SS C as shown by TLC. Elementary analysis and calculation from

the empirical formula C29H4QN706Br gave the following values:

C = 52.28% (52.57%), H = 6.16% (6.09%), N = 15.09% (14.80%) and Br = 11.85% (12.06% ).

Id. 2HBr. H-CHA-Arg-pNA

5.30 g (8 mmol) of compound 1c was treated with stirring with 32 ml of 2N HBr in glacial acetic acid for 40 min. at 20°C. The dipeptide derivative eventually dissolved during CO2 evolution. The reaction solution was added dropwise with vigorous stirring to 250 ml of absolute ether. This resulted in precipitation of 2HBr.H-CHA-

Arg pNA. The ether phase was suctioned off, after which the solid phase was washed four times with 100 ml portions of absolute ether to remove benzyl bromide which had formed as a by-product together with an excess of HBr and AcOH. The residue was dissolved in 50 mL of MeOH. The pH was adjusted to 4.5 using Et₂N, and the solution was concentrated to dryness in vacuo at 30°C. The resulting residue was dissolved in 50 ml of MeOH and purified on a column of "Sephadex LH-20" equilibrated with MeOH. The fraction of the MeOH eluate which was cleaved by treatment with trypsin to release p-nitroaniline was concentrated to dryness in vacuo at 30°C. The resulting residue was dried in a vacuum desiccator at 40°C over P2°5 to give 4.48 g (91.9% of theory) of amorphous compound 1d which was homogeneous in SS C. Elemental analysis and calculation from the empirical formula C21<H> 35<N>7<0>4<B>r gave the following results: C = 41.80% (41.39%), H = 5.86% (5.79%), N = 16.31% ,(16.09%) and Br = 25.85%

(26.23%).

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

3.05 g (5 mmol) of compound 1d was dissolved in 20 ml of freshly distilled DMF and the solution was cooled to -10°C. 0.70 ml

(5 mmol) Et^N was added to the solution with stirring. The formed Et^N.HBr was removed by filtration and washed with a small amount of cold DMF. 1.97 g (5.5 mmol) of Cbo-D-But.OpNP was added to the filtrate at -10°C with stirring. The reaction mixture was allowed to react for 2 to 3 hours without moisture, during which the temperature in the reaction solution gradually reached approx. 20°C. The solution was again cooled to -10°C and buffered with 0.35 ml (2.5 mmol).Et^N and allowed to react approx. 2 hours at -10°C and a further 3 hours at room temperature. This procedure was repeated with 0.35 ml Et^N, bg after 16 hours the reaction solution was concentrated to dryness in vacuo at 50 C. The residue was dissolved in 50 ml 50% AcOH and concentrated by gel filtration on a column of "Sephadex G -15" equilibrated with 50% AcOH. The main fraction of the AcOH eluate which was cleaved by treatment with trypsin releasing p-nitroaniline was concentrated to dryness in vacuo at 40°C. The residue was dissolved in 100 ml of MeOH and the solution was again concentrated to dryness. The resulting residue was treated in a vacuum desiccator at

60°C over P205 which gave 3.27g (87.5% of the theoretical)

of the amorphous compound 1e which was homogeneous in SS C as shown by TLC. Elemental analysis and calculation from the empirical formula C33H47<Ng0>7Br gave the following values: C = 52.88% (53.01%) , H = 6.40% (6.34%), N = 15.28% (14.99% ) and Br =10.53%

(10.69%).

lf. 2HBr. H-D-CHG-But-Arg-pNA

2.24 g (3 mmol) of compound 1e was treated with 12 ml of 2N HBr in glacial acetic acid for 40 min while stirring. at 20°C without humidity. The tripeptide derivative gradually resolved during decarboxylation

and C02 evolution. The reaction solution was added dropwise to 120 ml of absolute ether. This led to precipitation of 2HBr.H-D-But-»CHA-Arg-pNA. The ether phase was sucked off through a filter rod and then the solid phase was washed 4 times with portions of 50 ml of absolute ether. The resulting residue was dissolved in 40 ml of MeOH. The pH was adjusted to 4.5 using Et 3 N, and the solution was concentrated to dryness in vacuo at 30°C. The residue was dissolved in 30 ml of MeOH and purified on a column of "Sephadex LH-20" equilibrated with MeOH. The fraction of the MeOH eluate which was cleaved by treatment with trypsin to liberate p-nitroaniline was concentrated to dryness in vacuo at 30°C. For further purification, the prepurified residue was 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 was cleaved by treatment with trypsin to release p-nitroaniline was concentrated to dryness in vacuo at 40°C. The resulting residue was dried in a vacuum desiccator at 40°C over ^2Q5 to yield 1.71 g (82.1%)

of theory) amorphous compound 1f which was homogeneous in SS C as shown by TLC. Elementary analysis and calculation from summation

len C25H42N8°5Br2 ^a de *$ l9en& e values: C = 43.18% (43.24%),

H = 6.16% (6.10%), N = 16.27% (16.14%) and Br = 22.73% (23.01%).

The amino acid analysis confirmed the presence of the expected amino acids in the correct ratios:

Arg: 1.01 - CHA: 0.98 - D-But: 1.00.

Example 2

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

2a. BOC- Lys( f- Cbo)- pNA

38.05 g (0.1 mol) of dried oil of BOC-Lys(£-Cbo)-OH was dissolved in a 500 ml three-well flask in 150 ml of absolute HMPTA at 20°C without moisture. To this resulting solution was first added at room temperature a solution of 10.12 g (0.1 mol) Et3N in 25 ml HMPTA and then 24.62 g (0.15 mol), p-nitrophenyl isocyanate (50% excess) in portions, whereby strong C02 evolution took place each time. After a reaction time of 24 hours at 20°C, the main part of HMPTA was removed by distillation in vacuum. The residue was extracted several times with 2% NaHCO 3 solution and then treated with distilled H 2 O. This resulting residue was dried in vacuo at 40 C and then extracted several times with hot MeOH until the residue contained only the sparingly soluble by-product N,N'-bis(p-nitrophenylurea). The MeOH extracts were concentrated to 300 ml, during which , some impurities formed a flocculent precipitate. After filtration, the filtrate (330 ml) was purified on a column of "Sephadex LH-20" equilibrated with MeOH. The second fraction of the MeOH eluate was concentrated in vacuo at 30°C to a small volume , during which a needle-like substance crystallized. The crystals obtained were filtered off and washed in portions with 50 ml of ice-cold MeOH. After drying in a vacuum desiccator at 40°C above. P2O5 31.1 g (62.1 % of theoretical yield) crystalline compound 2a with m.p. which was homogeneous in SS A and B as shown by TLC The mother liquor gave a further yield of 5.8 g (11.6% of theory) of substance 2a with m.p. which was homogeneous in SS A and B as shown by TLC Elemental analysis and calculation from the sum formula C25<H>32<N>4°7 ^a de ^Øl9e nth values: C = 60.23% (59.99%), H = 6.50% (6.44%). and N = 11.38% (11.19%).

2b. CF3 COOH. H- Lys (E- Cbo)- pNA

25.03 g (50 mmol) of compound 2a was treated under vigorous stirring with 50 ml of freshly distilled anhydrous trifluoroacetic acid at 20°C for 60 min. in the absence of moisture. The BOC group was selectively cleaved during CO2 evolution and release of iso-butylene. The reaction solution was added dropwise with vigorous stirring to 750 ml of absolute ether, during which the CF3COOH.H-

Light (£-Cbo).-pNA is precipitated. The ether phase was sucked off through a filter rod. The solid phase was treated 4 times with 100 ml portions of absolute ether. The resulting residue was dissolved in 200 mL of MeOH. The pH was adjusted to 4.5 with Et^N and the solution was concentrated to dryness in vacuo at 30 C. The residue was dissolved in 200 ml of MeOH and purified on a column of "Sephadex LH-20" equilibrated with MeOH. The major fraction of the MeOH eluate which was homogeneous in SS C as shown by TLC was concentrated in vacuo at 30°C. The resulting residue was dried in a vacuum desiccator at 40°C over ^ 2°^ °9 gave 22.64 g (88.0% of theoretical yield amorphous compound 2b. Elemental analysis and calculation from the general formula C22H25N40?F3 gave the following values: C = 51.66% (51.36%1, H = 4.88%

(4.90%) and N = 11.08% (10.89%).

2 c. BOC- CHA- Lys(€- Cbo)- pNA

7.72 g (15 mmol) of compound 2b was dissolved in 50 ml of freshly distilled DMF and cooled to -10°C. 6.48 g (16.5 mmol) BOC-CHA-OpNP and 2.09 ml (15 mmol) Et^N were added to the solution with stirring. The mixture was allowed to react for 3 hours in the absence of moisture, whereby the reaction temperature gradually reached room temperature. The solution was again cooled to -10°C and buffered with 1.05 ml (7.5 mmol) Et 3 N. After a reaction time of 5 hours, this procedure was repeated with 1.05 ml of Et^N. After a reaction time of 16 hours at 20°C, the reaction solution was concentrated to dryness in vacuo at 50°C. The residue was dissolved in 150 ml of MeOH and purified by gel filtration on a column of "Sephadex LH-20" equilibrated with MeOH. The first fraction of the MeOH eluate which was homogeneous in SS A and B as shown by TLC was concentrated to dryness in vacuo at 30°C. After drying in a vacuum desiccator at 40°C over P2°5, 8.26 g (84.2% of theory) of compound 2c was obtained which was homogeneous in SS A and B as shown by TLC. Elemental analysis and calculation from the sum formula C34H47<N>5<0g>gave the following values: C = 63.05% (62.46%),

H = 7.35% (7.25%) and N = 10.98% (10.71%).

2d. CF3 COOH. H- CHA- Lys( £- Cb6)- pNA

3.27 g (5 mmol) of compound 2c was treated under vigorous stirring with 10 ml of freshly distilled anhydrous trifluoroacetic acid for 60 min. at 20°C in the absence of moisture. The reaction was added dropwise with vigorous stirring to 100 ml of absolute ether, whereby amorphous CF3COOH.H-CHA-Lys (C-Cbo)-pNA precipitated. The ether phase was suctioned off. The solid residue was washed 3 times with 30 ml portions of absolute ether. The resulting residue was dissolved in 50 mL of MeOH. The pH was adjusted to 4.5 using Et 3 N and the solution was concentrated to dryness in vacuo at 30°C. The residue was dissolved in 75 ml of MeOH and purified on a column of "Sephadex LH-20" equilibrated with MeOH. The major fraction of the MeOH eluate which was homogeneous in SS C as shown by TLC was concentrated in vacuo at 30°C. After drying the residue in a vacuum desiccator at 40°C and P2°5, 3.06 g (91.6% of theory) of amorphous compound 2d are obtained. Elemental analysis and calculation from the sum formula C3lH4oN5°8F3 ^a the following values: C = 56.08% (55.77%), H = 6.15%.(6.04%) and N = 11.01% (10, 49%)•

2nd. Cbd- D- But- CHA- Lys ( £- Cbo).- pNA

2.00 g (3 mmol) of compound 2d was dissolved in 15 ml of freshly distilled DMF cooled to -10°C. 1.36 g (3.3 mmol) Cbo-D-But-OpNP and 0.42 ml (3 mmol) Et3N were added to the solution while stirring. The mixture was allowed to react for 3 hours in the absence of moisture, during which the temperature gradually reached 20 C. The reaction solution was again cooled to -10°C and buffered with 0.21 ml

(1.5 mmol) Et 3 N. After a reaction time of 5 hours at -10°C, the temperature in the reaction mixture eventually reached room temperature. This procedure was repeated with 0.21 ml of Et3N during which the reaction took approx. 16 hours. The reaction mixture was concentrated to dryness in vacuo at 50°C and the residue was 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 was homogeneous in SS A and B as shown by TLC was concentrated to dryness in vacuo at 30 C. The residue was dried in a vacuum desiccator at 40 C over P^O^ to give 2.12 g (91, 4% of theoretical partially crystalline compound 2e.Elementary analysis and calculation from the sum formula

<C>41H52N6°9ga de f^ 1Sending values: C = 63.48% (63.71%), H = 6.82% (6.78%) and N = 10.99% (10.87%) .

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

1.55 g (2 mmol). compound 2e was treated with stirring with 12 ml of 2NHBr in glacial acetic acid for 40 min. and 20°C in the absence of moisture. The tripeptide derivative was gradually dissolved with simultaneous cleavage of the two protective groups Cbo and BOC and CC^ development. The reaction solution was added dropwise with vigorous stirring to 100 ml of absolute ether, whereby the 2HBr.H-D-But-CHA-Lys-pNA precipitated. The ether phase was sucked off after 30 minutes and the solid phase was washed 4 times with portions of 25 ml of absolute ether. The resulting residue was dissolved in 40 mL of MeOH. The pH was adjusted to 4.5 using Et^N and the solution was concentrated to dryness in vacuo at 30 C. The residue was dissolved in 30 ml of MeOH and purified on a column of "Sephadex LH-20" equilibrated with MeOH. The main fraction of the MeOH eluate which was cleaved by treatment with trypsin to liberate p-nitroaniline was concentrated to dryness in vacuo at 30°C. For further purification, the pre-purified product was dissolved in 25 ml of 33% AcOH and purified by gel filtration on a column of "Sephadex G-15" equilibrated with 33% AcOH. The fraction of the AcOH eluate which was cleaved by treatment with trypsin to liberate p-nitroaniline was concentrated to dryness in vacuo at 40°C. The resulting residue was dried in a vacuum desiccator at 40°C over P-jOj. and gave 0.99 g (74.3% of theory) of amorphous substance 2f which was homogeneous in SS C as shown by TLC. Elemental analysis and calculation from the sum formula C ^H^Ng*^*^ gave the following values: C = 44.75% (45.05%), H = 6.39% (6.35%), N = 12.67 % (12.61%) and Br = 23.68% (23.98%).

The amino acid analysis confirmed the presence of the expected amino acids in the correct ratios:

CHA: 0.98 - Light: 1.00 - D-But: 1.02.

Example 35

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

7.09 g (10 mmol) of 2HBr.H-D-Nval-CHA-Arg-pNA (prepared according to Example 38) was dissolved in 75 ml of 60% aqueous MeOH. The solution was applied to a column of "Amberlite" (Trademark) JRA-401

in acetate form. The column was eluted with 60% aqueous MeOH whereby HBr was replaced by AcOH by ion exchange. The eluate was concentrated to dryness in vacuo at 40°C. After drying

in a vacuum desiccator at 40°C over ?2Q5^iklc one 6.33 g of bromine-free 2AcOH.H-D-Nval-CHA-Arg-pNA (98.5% of theory).

According to this method, other salts with organic acids, e.g. formic acid, propionic acid, oxalic acid, tartaric acid, citric acid, lactic acid, benzoic acid, chlorobenzoic acid, salicylic acid or phthalic acid are produced from the above-mentioned tripeptide derivative.

An ion exchanger, e.g. "Amberlite" JRA-401 in hydrochloride form can be used and transferred into the desired acid salt form by transferring this ion exchanger into the basic OH form through treatment with caustic soda solution and then treating the basic ion exchanger with a solution of a 1:1 mixture of the desired organic acid and its sodium salt in 60% aqueous MeOH.

The quantitative enzyme determination using the tripeptide substrates according to the invention can be carried out as follows:

1. Determination of urinary kallikrein.

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

1.4 ml of distilled water heated to 37°C and 0.4 ml of centrifuged are mixed well in a plastic cuvette. For this mixture

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0.2 ml of a 2 x 10 molar aqueous substrate solution is added, and the components are mixed quickly. This mixture is incubated for exactly 15 min. 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 sample is used which consists of the

same ingredients, but with the glacial acetic acid added before the addition of the substrate to prevent the enzymatic reaction. The amount of colored R-NH^ which is formed photometrically or spectrophotometrically at 405 nm is then determined from the difference between the blank sample and the test sample. From the value obtained, the urinary kallikrein activity in urine was calculated according to the following formula:

Z^ OD = increase in optical density at 405 nm during 15 min. V = total volume of test mixture = 2.2 ml

1000 = transfer factor for transfer of U in mU

F = dilution factor of urine (2i

v = volume of the sample = 0.4 ml

= extinction coefficient divided by 1000 = 10.4

The calculation of the urinary kallikrein content in the urine can also be carried out by continuous measurement of the product R-NH^ (e.g. p-nitroaniline) which is formed. The method is described hereafter as used for measuring glandular kallikrein in saliva.

In addition to urinary lycraine, urine also contains urokinase as a protolytic enzyme which can also cleave substrates according to the present invention, albeit to a small extent.

In the determination method described above, the sum of the activities of urinary cabbage likrein and urokinase is measured. To obtain the exact value for urinary kallikrein activity, the urokinase activity must be subtracted. The latter can be determined in a comparison sample by adding 0.075 units of trypsin inhibitor (trypsin inhibitor from young cattle) per ml of buffer to completely inhibit urinary kallikrein activity and only measure urokinase activity.

2. Determination of glandular kallikrein in saliva:

0.5 ml of saliva is mixed with 2 ml of TRIS-imidazole buffer (ionic strength 1.0) and the mixture is incubated at 37<0>g for 5 min.

The incubate is centrifuged. A sample cuvette is filled with 1.5 ml of distilled water at 37°C and 0.25 ml of centrifuge is added. The ingredients are mixed well. Then 0.2 ml of

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a 2 x 10 molar aqueous substrate solution. The change in extinction at 405 nm is then monitored continuously for 5 to 10 min. by means of a registration. From the measured values for A OD per my. the kallikrein activity is calculated per ml of saliva

in mU using the following formula:

F = 5

V = 1.95

v = 0.25

1U (unit) = amount of enzyme that can cleave 1 µmole of substrate i

1 min. under optimal or otherwise defined conditions of pH, ionic strength, temperature and substrate concentrationv

In pancreatic saliva, pancreatic kallikrein is present mainly in the form of prekallikrein and can only be determined after activation, e.g. using trypsin. After activation of prekallikrein, the trypsin is inhibited using soybean trypsin inhibitor (SBTI). The kallikrein content in this activation mixture can be determined by one of the methods described above.

3. Determination of plasmin:

1.7 ml of TRIS-imidazole buffer (pH. 7.5, ionic strength 0.2). is mixed with 0.1 ml of a solution of plasmin in 25% glycerol at 37°C and the mixture is incubated at 37°C for a min.. 0.2 ml of an aqueous 2 x 10 3 molar substrate solution at 37°C is added to the mixture , and the ingredients are mixed quickly. The amount of the split product R-NH2 that is released from the substrate per time unit is then measured continuously. From the value determined per my. calculated the plasmin activity per ml of sample in mU from the following formula:

/^ E = amount of split product released per my.

V = total volume of sample mixture

v = volume of sample

£ = extinction coefficient divided by 1000

4. Determination of antiplasmin in human plasma:

0.1 ml of plasma diluted with TRIS-imidazole buffer in a ratio of 1:20 is mixed with 0.02 ml of a solution of 1.25 CU human plasmin (preparation from the company AB Kabi, Stockholm, Sweden), and 50 ATU hirudin (preparation from the company Pentapharm A.G. , Basel, Switzerland) per ml in 25% glycerol. The mixture is incubated for 9.0 seconds at 37°C. The incubation is mixed with 1.7 ml of trisimidazole buffer (pH 7.5, ionic strength 0.2). at 37°C and then with 0.2 ml of

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a 2 x 10 m61ar aqueous substrate solution. The amount of the split product R-NH^ total that is released from the substrate per time unit is then measured continuously. From the measured value, the remaining plasmin activity is calculated in the above-mentioned manner.

In the eh!blind test, the plasma is replaced with the corresponding amount of buffer, but otherwise the test is carried out in the manner described above. The measured plasmin activity corresponds to the amount of starting amine. The antiplasmin activity is calculated from the difference between the plasmin activity measured in the blank sample and the remaining plasmin activity measured in the sample using plasma according to the following formula:

F = dilution factor from plasma (20)

The substrates according to the invention can also be used for the determination of plasminogen in human plasma by transferring the plasminogen present in plasma using urokinase or streptokinase in a buffer system and measuring the amount of plasmin formed using one of the substrates according to the invention according to to the plasmin determination method described above. The amount of plasminogen originally present in the plasma is deduced from the value determined for plasmin as a molecular plasmin is formed from a molecular plasminogen upon activation.

The following table 4 shows the sensitivity of some of the substrates according to the invention to organ or gland kallikrein, plasmin and thrombin.

The measurement of the cleavage product R-NH2 formed by enzymatic hydrolysis of the substrate is based on the assumption that the cleavage product has a UV spectrum which differs from the substrate and which is shifted towards higher wavelengths. The absorption of the substrate at 405 nm is practically zero. p-Nitroaniline as a cleavage product shows an absorption maximum at 380 nm and a molar extinction coefficient of 13,200. At 405 nm, the extinction coefficient is only slightly lower, i.e. 9650. The degree of enzymatic hydrolysis of the substrate which is proportional to the amount of released p- nitroaniline can be determined by spectrophotometric measurement at 405 nm. Even in the presence of excess substrate, the measurement at 405 nm is not disturbed.

In the case of the substrates containing a 2-naphthylamino-, 4-methoxy-2-naphthylamino, 4-methyl-coumaryl-(71-amino or 1,3-di(methoxycarbonyl).-phenyl-(5 ).-amino group the quantity of the cleavage product R-NH2 is measured by fluorescence spectrophotometry. With a test system consisting of enzyme, buffer and substrate, the emitted light with lower energy is measured continuously at 400-470 nm after the formed fluorescent cleavage product has been excited by light with higher energy. The amount of cleavage product formed per unit of time is a measure of the existing enzyme activity. As indicated, a ;amol of cleavage product per minute corresponds to 1 enzyme unit based on a given substrate.

Claims (6)

lo Tripeptide derivative, characterized by the general formula H-D-X-Y-Z-R, where a) X represents a cyclohexylglycyl, cyclohexylalanyl or cyclohexyltyrosyl group, and Y represents an alanyl, α-aminobutyryl, norvalyl, leucyl, norleucyl or pipecoline group, or b) X represents a cyclohexylglycyl -, cyclohexylalanyl, cyclohexyltyrosyl or phenylglycyl group, and Y represents a phenylalanyl or phenylglycyl group, or c) X represents an alanyl, α-aminobutyryl, valyl, norvalyl, leucyl or norleucyl group, and Y represents a cyclohexylalanyl, cyclohexyltyrosyl or phenylglycyl group, or d) X represents a phenylalanyl or phenylglycyl group and Y represents a cyclohexylalanyl or cyclohexyltyrosyl group, Z represents an arginyl or lysyl group and R represents p-nitrophenylamino, 2-naphthylamino, 4-methoxy-2-naphthylamino, 4-methyl-coumaryl-7-amino, 1, 3-di(methoxy-carbonyl)phenyl-5-amino, quinonyl-5-amino or 8-nitro-quinonyl-5-amino and salts thereof with acids, with the proviso that the tripeptide derivative cannot be H-D-CHG-Phe-Lys -pNA, H-D-Val-CHA-Arg-MCA, H-D-CHG-Phe-Lys-MCA, H-D-Val-CHA-Arg-DPA, H-D-CHG-Phe-Lys-DPA, H-D-Val-CHA-Arg -2-NA, H-D-CHG-Phe-Lys-2-NA, H-D-Val-CHA-Arg-4-MeO-2-NA, H-D-CHG-Phe-Lys-4-MeO-2-NA, H-D-CHG-Leu- Arg-pNA, H-D-Val-CHÅ-Arg-pNA, H-D-Val-CHA-Lys-pNA, H-D-CHG-Phe-Arg-pNA, H-D-CHT-Phe-Arg-pNA, H-D-Ph<1- >Gly-CHA-Arg-pNA, H-D-CHG-Leu-Lys-pNA or H-D-CHT-Pip-Arg-pNA. 2. Tripeptide derivative according to claim 1, characterized in that the dipeptide fragment bound to Z is a CHG-Ala-, CHG-But-, CHG-Norval-, CHG-Leu-, CHG-Norleu-, CHG-Pip-, CHA-Ala-, CHA-But, CHA-Norval, CHA-Leu, CHA-Norleu, CHA-Pip, HCA-But, HCA-Leu or HCA-Pip group. 3. Tripeptide derivative according to claim 1, characterized in that the dipeptide fragment bound to Z is a CHG-Phe-, CHG-Ph'Gly-, CHA-Phe-, CHA-Ph'Gly-, HCA-Phe-, HCA-Ph'Gly- , or Ph'Gly-Phe group. 4. Tripeptide derivative according to claim 1, characterized in that the dipeptide fragment bound to Z is an Ala-CHA-, Ala-Ph'Gly-, But-CHA-, Val-CHG-, Val-CHA-, Val-HCA-, Norval-CHA -, Norval-Ph'Gly, Leu-CHA, Leu-Ph'Gly, Norleu-CHA or Ile-CHA group. 5. Tripeptide derivative according to claim 1, characterized in that the dipeptide fragment bound to Z is a Phe-CHA-, Phe-CHG-, Ph'Gly-CHA-, Ph'Gly-CHG-, Ph'Gly-HCA-, CHG-CHA- or CHG-CHG group. 6. Use of the tripeptide derivatives according to claim 1 for quantitative detection of proteolytic enzymes of enzyme class 3.4.21.