DK159458B - TRIPEPTID-P-NITROANILIDES AND ITS USE FOR QUANTITATIVE ANALYSIS FOR PROTEOLYTIC ENZYMES - Google Patents

Description

in

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The present invention relates to novel tripeptide derivatives useful as substrates for quantitative analysis for proteolytic enzymes from the enzyme class E.C. 3.4.21, in particular thrombin and plasmin.

More specifically, the present invention relates to novel chromogenic substrates which have high sensitivity to certain proteolytic enzymes of the enzyme class E.C. 3.4.21., Especially thrombin and plasmin, and are therefore useful as substrates for quantitative analysis for these enzymes. These substrates are tripeptide derivatives of the general formula I

H-D-X-Y-Arg - p-nitroanilide I

wherein X represents cyclohexylglycyl (CHG), cyclohexylalanyl (CHA) or cyclohexyltyrosyl (CHT) and Y represents alanyl, α-aminobutyryl (But), or norvalyl, and their salts with acids.

The tripeptide derivatives of formula I are highly 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 acid, hydrochloric acid, sulfuric acid, phosphoric acid, 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; benzoic or phthalic acid. The nature of the acid is not critical as 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 by the action of certain proteolytic enzymes of the enzyme class 3.4.21. (cf. "Enzyme Nomenclature", Elsevier Scientific Publishing Company, Amsterdam 1973, page 238 et seq.), in particular thrombin and plasmin, and as a result a colored or fluorescent cleavage product which is p-nitroaniline is formed, the amount of which can be measured by photometric , spectrophotometric, fluorescence spectrophotometric or electrochemical

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2 methods. The novel substrates are therefore useful for quantitative analysis for proteolytic enzymes from the enzyme class E.C.

3.4.21., Which cleaves natural peptide chains on the carboxy side of arginine, e.g., plasmin and thrombin, as well as their inhibitors and proenzymes.

The invention therefore also relates to a method of quantitative analysis for proteolytic enzymes from the enzyme class E.C. 3.4.21., Which cleaves natural peptide chains on the carboxy side of arginine, especially thrombin and plasmin, in media containing the enzymes. 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 p-nitroaniline formed by the hydrolytic action of the enzyme on the tripeptide derivative is measured by photometric, spectrophotometric , fluorescence spectrophotometric or electrochemical methods.

15 Tripeptide derivatives which can be used as substrates for the determination of enzymes of the enzyme class E.C.3.4.21 such as plasmin and thrombin are described in Danish Patent Application No. 3127/76, Danish Patent Application No. 3126/76 and Danish Patent Application No. 2400/73. However, it is neither suggested nor shown that the 20 tripeptide derivatives described in these patent applications are cleaved by both plasmin and thrombin at an excellent and useful cleavage rate, as is the case for the compounds of the present invention. The tripeptide derivatives described in the above-mentioned Danish applications are also structurally different from the tripeptide derivatives according to the invention. Thus, the tripeptide derivatives known from Danish patent application 3127/76 are structurally different from the tripeptide derivatives of the present invention in that their N-terminal amino acid either does not contain side chains or contains a straight or branched alkyl group of 1-4 carbon atoms. In addition, the N-terminal 30 amino group may also be a cyclic amino acid. In contrast, the N-terminal amino group of the tripeptide derivatives of the present invention contains a sterically bulky and hydrophobic side chain containing 6-7 carbon atoms and carrying a cyclohexyl ring.

The tripeptide derivatives described in Danish patent application no.

35 3126/76, contains as a N-terminal amino group a D-amino acid whose

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3 side chain contains an aromatic phenyl ring and 7 carbon atoms. These tripeptide derivatives also contain as a middle amino acid a 4-6 membered cyclic amino acid. In contrast, the N-terminal amino acid of the tripeptide derivatives of the present invention, as mentioned above, contains a side chain containing 6-7 carbon atoms and a non-aromatic cyclohexyl ring. In addition, the middle amino acid has an open side chain which is constituted by a straight chain alkyl group having 1-3 carbon atoms.

The tripeptide derivatives described in Danish patent application no.

10 2400/73 differs from the tripeptide derivatives of the present invention in that their N-terminal amino acids have a side chain which contains a phenyl ring and is therefore of an aromatic nature. This is in contrast to the tripeptide derivatives of the present invention, whose N-terminal amino group, as mentioned above, has a side chain containing a cyclohexyl ring and thus having a highly hydrophobic and cycloaliphatic character.

The tripeptide derivatives of formula I can be prepared by the methods described below: 1) The chromogenic group p-nitroanilide (pNA) is attached to the carboxy group of the C-terminal arginine, wherein the α-amino group is protected by a protecting group, e.g. a carbobenzoxy or t-butoxycarbonyl group. in the guanidyl group for arginine is protected by protonation, for example with HCl, nitration or tosylation. The C-terminal pNA group also serves as a protecting group during the stepwise synthesis of the peptide chain. The other protecting groups may 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 pNA-NH group (cf., e.g., Miklos Bodansky et al., 30 "Peptide Synthesis", Interscience Publishers, 1966, pp. 163 - 165).

2) First, the peptide chain (according to Bodansky et al., Loc. Cit.) Is synthesized, however, where the C-terminal carboxyl group in arginine is protected using a conventional ester group, for example a methoxy, ethoxy or benzyloxy group. The ester groups can be removed by alkaline hydroxide.

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4 except the tert-butoxy group, which must be selectively cleaved by trifluoroacetic acid. If the S-guanidyl group in arginine is protonized, this ester group is enzymatically removed by trypsin, with no racemization taking place. Then the chromogenic pNA-NH group is attached. When the β-guanidino group in arginine is protected with a nitro or tosyl group and the N-terminal α-amino group in the tripeptide derivative is protected with a carbobenzoxy group or a p-methyl, p-methoxy or p-chlorobenzyloxycarbonyl group or a tert-butoxy group, all protection 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 of the said protecting groups on the amino and δ-guanidino groups. The removal can also be accomplished by treatment with 2N hydrochloric acid in glacial acetic acid at room temperature if the protected tripeptide derivative does not contain 15 nitro or tosyl protecting groups.

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

Assays of eluates and products prepared according to the Examples are performed by thin layer chromatography using 20 glass plates coated with silica gel (Merck, F 254). The thin layer chromatograms are developed using the following solvent systems: A Chloroform / methanol (9: 1). ~ B n-Propanol / ethyl acetate / water (7: 1: 2).

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

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5

The following abbreviations are used:

AcOH = acetic acid

Ala = alanine

Arg = arginine BOG = tert.butoxycarbonyl

But = α-amino butyric acid

Cbo = carbobenzoxy CHA = cyclohexylalanine CHG = cyclohexylglycine CHT = cyclohexyl tyrosine = p-hydroxycyclohexylalanine DMF = dimethylformamide TLC = thin-layer chromatography ΕίβΝ = triethylamine HMPTA = phosphoric acid Ν, f, f isoleucine

Leu = leucine SS = solvent system / s

MeOH = methanol

Nleu = norleucine 20 Nval = norvaline

OpNP = p-nitrophenoxy

Phe = phenylalanine pH'Gly = phenylglycine

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

Fall = fall in

Unless otherwise indicated, the amino acids in the peptide chains are L-shaped.

EXAMPLE 1

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6 H-D-CHG-Ala-Arg pNA. 2HBr.

la. Cbo-Arg-pNA.HC1.

In a 250 ml three-neck flask, dissolve 16.0 g (47.0 millimoles) of Cbo-Arg-5OH.HCl, dried in vacuo over P2O5, in 90 ml of absolute HMPTA at 20 ° C while keeping the atmosphere in the flask moist . To the resulting solution is added at room temperature first a solution of 4.74 g (47.0 millimoles) of ΝίβΝ 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 C20 H25 N6 O5 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 results in the 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 the solution is evaporated to dryness in vacuo at 30 ° C. The resultant

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7 product is dissolved in 1 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 theoretical) of amorphous compound 1b is obtained, which is homogeneous in solvent system C as shown by thin layer chromatography.

Analysis:

Calcd for ^ 2 ^ 20 ^^ 3 ^ 2 C 31.60 H 4.42 N 18.43 Br 35.03.

Found: C 31.15 H 4.35 N 18.84 Br 34.81.

lc. Cbo-Ala-Arg-pNA.HBr.

Dissolve 2.28 g (5 millimoles) of compound 1b in 30 ml of freshly distilled DMF and cool the solution to -10 ° C. 0.70 ml (5 millimoles) of Et3N is added to the solution with stirring. The Et3N.HBr formed is removed by filtration and washed with a small amount of cold dimethylformamide. 1.89 g (5.5 millimoles) of Cbo-Ala-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, thereby gradually reaching the temperature of the reaction solution. 20 ° C. The solution is again cooled to -10 ° C, buffered with 0.35 ml (2.5 millimoles) of Et3N and allowed to react for 2 hours at -10 ° C and for approx. 3 hours at room temperature. This procedure is repeated with 0.35 ml ΕίβΝ and after 20 16 hours the reaction solution is evaporated to dryness in vacuo at 50 ° C.

The residue is dissolved in 40 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 trypsin treatment with release of p-nitroaniline, is evaporated to dryness in vacuo at 40 ° C.

The residue is dissolved in 75 ml of MeOH and evaporated again to dryness. The resulting residue is dried in a vacuum desiccator at 60 ° C over P 2 O 5 to give 2.57 g (88.5% of theory) of the amorphous compound 1c, which is homogeneous in solvent system C as shown by TLC.

Analysis:

Calcd for C 23 H 30 N 7 O 6 Br: C 47.59 H 5.21 N 16.89 Br 13.77.

Found: C 47.19 H 5.22 N 17.18 Br 13.60.

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8 ld. 2HBr.H-Ala-Arg-pNA.

1.74 g (3 millimoles) of compound 1c is treated with stirring with 12 ml of 2N HBr in glacial acetic acid for 40 minutes at 20 ° C. The peptide derivative dissolves gradually during C0 evolution. The reaction solution is added dropwise with vigorous stirring to 100 ml of absolute ether. This results in the precipitation of 2HBr.H-Ala-Arg pNA. The etheric phase is suctioned off and the solid phase is washed four times with 50 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 20 ml of MeOH. The pH 10 is adjusted to 4.5 using Et3N and the solution is evaporated to dryness in vacuo at 30 ° C. The resulting residue is dissolved in 25 mL of MeOH and purified on a column of "Sephadex LH-20" ® equilibrated with MeOH. The fraction of the MeOH eluate which is cleaved by treatment with trypsin with release of p-nitroaniline is evaporated to dryness in vacuo at 30 ° C. The resulting residue is dried in a vacuum exchanger at 40 ° C over P 2 O 5 to give 1.44 g (90.8% of theory) of amorphous compound ld, which is homogeneous in solvent system C.

Analysis:

Calcd. For C 15 H 25 N 7 O 4 Br: C 34.17 H 4.78 N 18.60 Br 30.31.

Found: C, 34.01; H, 4.76; N, 18.85; Br, 29.88.

scythe. Cbo-D-CHG-Ala-Arg-pNA.HBr.

Dissolve 0.78 g (1.5 millimoles) of compound 1d in 8 ml of freshly distilled DMF and cool the solution to -10 ° C. 0.21 ml (1.5 millimoles)

EtjN is added to the solution with stirring. The Et3N.Hbr 25 formed is removed by filtration and washed with a small amount of cold DMF. 0.68 g (1.65 millimoles) of Cbo-D-GHG.OpNP is added at -10 ° C 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 ° G. The solution is again cooled to -10 ° G, buffered with 0.105 ml \ 30 (0.75 millimole) of Et3N and allowed to react for approx. 2 hours at -10 ° G and for another 3 hours at room temperature. This procedure is repeated with 0.105 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 15 ml of 50% AcOH and

ÆV

is purified by gel filtration on a column of "Sephadex G-15" equiv.

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9 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 40 ml of MeOH and the solution is again evaporated to dryness. The resulting residue 5 is dried in a vacuum desiccator at 60 ° C over P2 ° 5 to give 936 mg (86.7% of theory) of the amorphous compound 1e, which is homogeneous in solvent system C as shown by TLC.

Analysis:

Calcd for CsiHz ^ NgOyBr: C 51.74 H 6.02 N 15.57 Br 11.10.

Found: C 51.66 H 6.06 N 15.80 Br 10.96.

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

0.72 g (1 millimole) of compound le is treated with stirring with 4 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 CO 2 development. The reaction solution is added dropwise with vigorous stirring to 40 ml of absolute ether. This results in the precipitation of 2HBr.H-D-CHG-Ala-Arg pNA. The etheric phase is aspirated through a filter rod and then the solid phase is washed four times with 15 ml portions of absolute ether. The resulting residue is dissolved in 15 ml of MeOH. The pH-20 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 trypsin with release of p-nitroaniline is evaporated to dryness in vacuo at 25 ° C. For further purification, the purified residue is dissolved in 10 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 over P 2 O 5 to give 556 mg (83.4% of theory) of amorphous compound 1f, which is homogeneous in solvent system C as shown by TLC.

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Analysis:

Calcd for 023Η38Ν805Β: C 41.45 H 5.75 N 16.82 Br 23.98.

Found: C 41.66 H 5.74 N 17.09 Br 23.90.

The amino acid analysis confirms the presence of the expected amino-5 acids in the correct ratios: D-CHG: 0.96 - Ala: 1.00 - Arg: 0.99.

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.

Table I

Forward Analysis

Starting Procedure Found Calculated% 15 Ways Way _

Eczema (mmol) (ex) Amino acid analysis final product Yield% 2 2HBr.HD-CHA-2e (1 mmol) (lf) 88.1 C 42.54 42.36 Ala-Arg-pNA 2N HBr / AcOH H 5.98 5.93 C24H40N8 ° 5Br2 N 16'60 16'47

Br 23.19 23.49 ...... - CHA: Ala: Arg - .......... 0.98: 1.00: 1.00 3 2HBr.H-D-CHA-3e (0.5 mmol) (lf) 90.2 C 43.90 44.08

Nval Arg Arg pNA 2N HBr / AcOH H 6.32 6.26 C26H44N8 ° 5Br2 N 16> 18 l5> 82

Br 22.39 22.56 CHA: Nval: Arg 30 0.97: 1.00: 0.99

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Table I continued

Forward Analysis

Initial Found Found Calculated% by way _ 5 Eczema (mmol) (ex) Amino acid analysis final product Yield% 4 2HBr.HD-CHG-4e (0.5 mmol) (lf) 88.5 C 43.20 43 , 24

Nval Arg Arg pNA 2N HBr / AcOH H 6.15 6.10 C25H42N8 ° 5Br2 N 16.36 16.14

Br 22.78 23.01 CHG: Nval: Arg 0.96: 1.00: 1.01 5 2HBr.HD-CHG-5e (1 mmol) (lf) 84.7 C 42.18 42.36 But -Arg-pNA 2N HBr / AcOH H 6.00 5.93 C24H40N8 ° 5Br2 N 16 * 52 16> 47

Br 23.18 23.49 CHG: But: Arg 0.97: 0.99: 1.00 20 6 2HBr.H-D-CHA- 6e (1 mmol) (lf) 88.5 C 42.96 43.24

But-Arg-pNA 2N HBr / AcOH H 6.12 6.10 C25H42N8 ° 5Br2 N 16> 25 16 · 14

Br 22.69 23.01 CHA: But: Arg 25 0.98: 0.98: 1.00 7 2HBr.H-D-CHT-7e (1 mmol) (lf) 82.3 C 41.89 42.26

But-Arg pNA 2N HBr / AcOH H 6.01 5.96 C25H42N8 ° 6Br2 N i5'96 i5> 77

Br 22.15 22.49 CHT: But: Arg 0.96: 0.99: 1.00 8 2HBr.H-D-CHT-8e (1 mmol) (lf) 82.7 C 41.61 41.39

Ala-Arg pNA 2N HBr / AcOH H 5.82 5.99 C24H40N8 ° 6Br2 N 16> 29 16'09 Br Br 22.75 22.95 CHT: Ala: Arg 0.97: 1.00: 0.98

Table II

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Flow

Starting Procedure 5 Eczema Dipeptide Products (Ex.) Analysis Pell Precursor (mmol) Yield% Found% Calculated% 3c Cbo-Nval-Arg-lb (5 mmol) (lc) 90.8 C 49.13 49.35 pNA.HBr Cbo-Nval-OpNP H 5.69 5.63 C25H34N706Br (5.5 mmol) N 16.38 16.11

Br 13.00 13.13 3d 2HBr.H-Nval-3c (3 mmol) (ld) 96.0 C 36.4'5 36.77

Arg-pNA 2N HBr / AcOH H 5.30 5.26 C17H29N7 ° 4Br2 N 18.01 17.66 Br 28.55 28.78 5c Cbo-But-Arg-lb (5 mmol) (lc) 91.6 C 48.09 48.49 pNA.HBr Cbo-But-OpNP H 5.46 5.43 C24H32N7 ° 6Br (5> 5 ππη ° 1) N 16.83 16.49

Br 13.22 13.44 5d 2HBr.Η-But-5c (3 mmol) (ld) 94.2 C 35.50 35.51

Arg-pNA 2N HBr / AcOH H 5.08 5.03 C16H27N7 ° 4Br2 N 18.40 18.12

Br, 29.08, 29.53

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Table III

Ex-Procedure Sem-Tripeptide Products (Ex.) Analysis 5 µl Precursor (nmol) Yield% Found% Calculated% 2e Cbo-D-CHA-Ala-ld (1.5 nmol) (le) 88.4 C 52.09 52.39

Arg-pNA.HBr Cbo-D-CHA-H 6.22 6.18 C32H45N8 ° 7Br 0pNP (1) 65 ^ 1 ° 1) N 15.55 15.27 10 Br 10.70 10.89 3rd Gbo-D -CHA- 3d (1 mmol) (le) 86.0 C 53.48 53.61

Nval-Arg-Cbo-D-CHA-H 6.46 6.48 pNA.HBr OpNP (1.1 nmol) N 14.80 14.71 C34H49NS ° 7Br Br 10> 31 10'49 15 4th Cbo-D CHG-3d (1 nmol) (le) 88.8 C 52.88 53.01

Nval-Arg-Cbo-D-CHG-H 6.35 6.34 pNA.HBr OpNP (1.1 mmol) N 15.18 14.99 C33H47N807Br Br 10.49 10.69 5th Cbo-D-CHG-But 5d (2 mmol) (le) 84.7 C 52.19 52.39 Arg-pNA.Hbr Cbo-D-CHG-H 6.17 6.18 C32H45N8 ° 7Br ° pNP (2) 2 N 15, 15.27

Br 10.70 10.89 6th Cbo-D-CHA-But-5d (2 mmol) (le) 80.6 C 52.87 53.01

Arg-pNA.HBr Cbo-D-CHA-H 6.41 6.34 C33H47Ng07Br OpNP (2.2 mmol) N 15.17 14.99

Br 10.55 10.69 7th Cbo-D-CHT-But-5d (2 mmol) (le) 83.3 C 51.72 51.90

Arg-pNA.HBr Cbo-D-CHT-H 6.25 6.20 C33H47N8 ° 8Br 0pNP (2) 2 nunol) N 14.88 14.67 Br 10.37 10.46 8e Cbo-D-CHT- Alde (1.5 mmol) (le) 79.4 C 51.01 61.27

Arg-pNA.HBr Cbo-D-CHT-H 6.09 6.05 C32H45N8 ° sBr ° pNP <1.65 ^ 11 ° 1) N 15) 10 14) 95

Br 10.48 10.66 Example 9

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14 2AcOH.H-D-CHG-Ala-Arg-pNA.

6.66 g (10 millimoles) of 2HBr.H-D-CHG-Ala-Arg pNA (prepared according to Example 1) are dissolved in 75 ml of 60% aqueous MeOH. The solution is passed on 5 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 P2O5, 6.15 g of bromide-free 2AcOH.H-D-CHG-Ala-Arg pNA (98.5% of theory) are obtained.

In this process, other salts may be prepared with organic acids, for example formic acid, propionic acid, oxalic acid, tartaric acid, citric acid, lactic acid, benzoic acid, chlorobenzoic acid, salicylic acid or phthalic acid, from the above-mentioned tripeptide derivative. An ion exchanger, e.g., "Amber-lite" JRA-401 in hydrochloride form, can be used to convert to the desired acid salt form by converting the ioribter to the basic OH form by treatment with sodium hydroxide solution and then treating the basic ion exchanger with a solution of 1 mixture of the excess organic acid and its sodium salt in 60% aqueous MeOH.

Thrombin analysis can be performed as follows: 0.25 ml of thrombin solution (0.56 NIH units of human thrombin per ml) is added to 2.0 ml of Tris-imidazole buffer (pH 8.4, ionic strength 0.15) .

The mixture is pre-incubated for 2 minutes at 37 ° C. Then 0.25 ml of aqueous substrate solution (2 μιηοΐ of a substrate according to the invention) is added to the preincubated mixture at 37 ° C. The absorbance rise is measured spectrophotometrically at 405 nm and is continuously monitored using a measuring equipment. The results obtained are given in Table IV.

The amount of cleavage product formed is a measure of the substrate's availability for thrombin. When calculating the amount (nanomol) of 30 p-nitroaniline formed per per minute, a molar extinction coefficient of 10,000 is used instead of 9,620 for convenience. This has no effect on the ratio of the availability of thrombin to the various substrates.

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Analysis for plasmin can be carried out as follows: 1.7 ml of tris-imidazole buffer (pH 7.5, ionic strength 0.2) are 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 1 minute. 0.2 ml of an aqueous 2 x 10 6 molar substrate solution is added to the mixture at 37 ° C and the ingredients are quickly mixed. The amount of the cleavage product p-nitroanilide released from the substrate per unit of time is then continuously measured.

From the value determined per Plasma activity per minute is calculated per minute. ml sample in mU based on the following formula: 10 ΔΕ / min. x V x 1000 __ = mU / ml of sample v x e ΔΕ = amount of cleavage product released per minute 10V = total volume of test mixture 15v - sample volume e = extinction coefficient divided by 1000

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Table IV

Activity of human NIH thrombin and human plasmin measured by the tripeptide β-nitroanilides of the invention at constant substrate and enzyme concentration. In comparison, the corresponding values 5 for the known tripeptide derivatives are 2HCl.HD-Phe-Pip-Arg-pNA (Compound VII in Danish Patent Application No. 3126/76) and 2HCl.HD-Pro-Phe-Arg-pNA (Compound XL in Danish patent application no. 3127/76) also stated. Substrate concentration: 2 x 10 'molar.

10 Tripeptide- Amount in nanomoles of p-nitroaniline, released in derivatives over 1 minute by 1 NIH unit of human thrombin and 1 CU unit of human plasmin, respectively

Substrate of the Invention Prepared in Ex. Human Thrombin Human Plasmid 1 158 2 163.5 20 3 97.1 574 4 67.4 488 5 102.9 439 6 108.9 478 7 114.2 463 25 8 156.4

Known Substrates VII 120.2 XL 451 30 NIH unit is a standard thrombin unit defined by the US National Institute of Health CU unit is a standard plasmin unit, cf. Commitée on

Thrombolytic Agents in Recommendations on Units, 35 published in Thrombosis DIATH, Haemorrhag'ica HEAM0RRH, vol. 30, p. 259, 1969.

Claims (5)

A tripeptide derivative of the general formula I HDXY-Arg - p-nitroanilide I wherein X represents a cyclohexylglycyl (CHG), cyclohexylalanyl (CHA) or cyclohexyltyrosyl group (CHT), and Y represents an alanyl, α-aminobutyryl - (But) or norvalyl group, and their salts with acids. Tripeptide derivatives according to claim 1, characterized in that the dipeptide fragment attached to Arg is CHG-Ala, CHG-But, CHG-Nval, CHA-Ala, CHA-But, CHA-Nval, or CHT-But. Tripeptide derivatives according to claim 1 or 2, characterized in that they are protonated with a mineral acid, for example hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid, or an organic acid, for example 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, core-substituted aromatic acids such as toluene acids, chloro or bromobenzoic acids, methoxybenzoic acids or aminobenzoic acids or phthalic acid. 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, 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 p-nitroaniline formed by the hydrolytic action of the enzyme on the tripeptide derivative is measured by photometric, spectrophotometric, fluorescence spectrophotometric or electrochemical methods. Method according to claim 4, characterized in that blood or blood plasma thrombin is analyzed.