EP0000063B1

EP0000063B1 - Dipeptide derivatives of 7-(n-alpha-substituted or non-substituted x-arginyl)-amino-4-methyl-coumarin

Info

Publication number: EP0000063B1
Application number: EP78100111A
Authority: EP
Inventors: Shumpei Sakakibara
Original assignee: Ajinomoto Co Inc
Current assignee: Ajinomoto Co Inc
Priority date: 1977-06-06
Filing date: 1978-06-06
Publication date: 1981-06-10
Also published as: US4215047A; EP0000063A1; DE2860749D1

Description

This invention relates to peptide derivatives, more particularly 7-(N"-substituted or non- substituted-X-arginyl)-amino-4-methyl-coumarin wherein X is a residue of an amino acid selected from the group of glycine, phenylalanine and proline, and to acid salts thereof, which are useful as fluorogenic substrates for determining enzymatic activities.
In order to determine enzymatic activities, a method is usually employed which comprises reacting a material which is specifically acted upon by an enzyme with the enzyme and comparing the state of the material before reaction with that after reaction.
As materials for the stated purpose, materials occuring in nature can be employed, but synthesized ones are suitably employed in view of mass production and production in pure form. Therefore, compounds which can be substrates for all kinds of enzymes are strongly desired. Such compounds can act as antimetabolites in a living body, and thus it is expected that such compounds can be used as medicines.
This invention responds to such need and provides synthetic substrates which show specificity to enzymes such as Trypsin and Thrombin.
Compounds of the present invention are novel peptide derivatives, which are not described in any reference, more particularly 7-(N^*-substituted or non-substituted-X-arginyl)-amino-4-methyl coumarin wherein X is a residue of an amino acid selected from the group of glycine, phenylalanine and proline, and acid salts thereof.

(1) When X is the residue of glycine, the compounds are represented by the following formula:
and salts thereof, wherein R, is a radical selected from the group consisting of a hydrogen atom and prolyl, glutamyl, seryl leucyl, isoleucylglutamyl, valylseryl and valylleucyl groups.

When R, is a hydrogen atom, the compound is 7-(glycylarginyl)-amino-4-methylcoumarin. When R, is a prolyl group, the compound is 7-(prolylglycylarginyl)-amino-4-methylcoumarin. When R₁ is a glutamyl group, the compound is 7-(glutamylglycylarginyl)-amino-4-methylcoumarin. When R, is a seryl group, the compound is 7-(serylglycylarginyl)-amino-4-methylcoumarin. When R, is a leucyl group, the compound is 7-(leucylglycylarginyl)-amino-4-methyl-coumarin. When R, is a glutamyl group carrying an isoleucyl group in the N^α-position thereof, namely an isoleucylglutamyl group, the compound is 7-(isoleucylglutamylglycylarginyl)-amino-4.-methylcoumarin. When R, is a seryl group having a valyl group in the N"-position thereof, namely a valylseryl group, the compound is 7-(valylserylglycyl- arginyl)-amino-4-methylcoumarin. When R, is a leucyl group having a valyl group in the N^α-position thereof, namely valylleucyl group, the compound is 7-(valylleucylglycylarginyl)-amino-4-methylcoumarin.
(2) When X is the residue of phenylalanine, the compounds of the present invention are represented by the following formula:
and salts thereof, wherein R₂ is a radical selected from the group consisting of a hydrogen atom and a prolyl group. When R₂ is a hydrogen atom, the compound is 7-(phenylalanylarginyl)-amino-4-methylcoumarin. When R₂ is a prolyl group, the compound is 7-(prolylphenylalanylarginyl)-amino-4-methylcoumarin.
(3) When X is the residue of proline, the compounds of the present invention are represented by the following formula:
and salts thereof, wherein R₃ is a radical selected from the group consisting of a hydrogen atom and a valyl group.
When R₃ is a hydrogen atom, the compound is 7-(prolylarginyl)-amino-4-methylcoumarin. When R₃ is a valyl group, the compound is 7-(valylprolylarginyl)-amino-4-methylcoumarin.
N"-amino and imino groups in such compounds may be protected by conventional protecting grqups for peptides, such as an acyl group (for example, acetyl group and benzoyl group), carbobenzoxy group, tert-alkyloxycarbonyl group, tosyl group and glutaryl group.
In case of the compounds having a serine residue, the hydroxy group thereof may be protected by protecting groups which are used in the conventional method for synthesizing peptides, such as alkyl groups and benzyl groups.
In case of the compounds having a glutamic acid residue, the y-carboxyl group thereof may be protected by an ester group with an alcohol such as benzyl alcohol.
The guanidino group of arginine which constitutes such compounds may be protected by N-guanidino-protecting groups which are used in the conventional method for synthesizing peptides, such as nitro groups, tosyl groups and p-methoxy-benzenesulfonyl groups, and by adding a proton, as in the form of an acid.
The compounds of the present invention can be produced by conventional methods for synthesizing peptides using 7-arginyl-amino-4-methylcoumarin. For example, 7-(glycylarginyl)-amino-4-methylcoumarin can be produced by reacting glycine whose amino group has been protected, with 7-arginylamino-4-methylcoumarin in the presence of condensing agents such as dicyclohexyl-' carbodiimide (DCCI), or by reacting an active ester of glycine whose amino group has been protected; with 7-arginylamino-4-methylcoumarin, and then removing the protecting-group of the amino group therein. By reacting this compound with glutamic acid whose amino group and y-carboxyl group are protected and then removing protecting-groups from both the amino group and y-carboxyl group of thus obtained compound, 7-(glutamylglycylarginyl)-amino-4-methylcoumarin can be obtained. 7-(isoleucyl-glutamylglycylarginyl)-amino-4-methylcoumarin can be produced by reacting 7-(glutamyl- glycylarginyl)-amino-4-methylcoumarin as produced above, with an active ester of isoleucine whose amino group has been protected, an then treating such reacted material in the same manner as above. 7-(prolylglycylarginyl)-amino-4-methylcoumarin can be produced by reacting 7-(glycylarginyl)-amino-4-methylcoumarin obtained above with proline whose imino group has been protected and then removing the protecting group from the thus obtained compound.
These condensing reactions can desirably be operated in a suitable solvent such as dimethylformamide (DMF), dimethylsulfoxide, water and mixtures thereof.
It is preferable that the component having the carboxyl group which reacts with the component having the amino group, is in the active ester form. Suitable examples of the active esters are N-hydroxysuccinimide ester and p-nitrophenyl ester. Reactions using active ester can be operated sufficiently at room temperature, but can be accelerated by heating, if necessary.
After completing the reaction, the reaction mixture is concentrated to yield a solid material and the material is purified by column chromatography and then lyophilized. The compounds thus obtained are usually white powders and are decomposed slowly by heating at more than 140°C.
As regards the compounds having protecting groups in amino or imino groups thereof, such protecting-groups can be removed by conventional methods or synthesizing peptides. For example, the carbobenzoxy group can be removed by catalytic hydrogenation in a solvent such as alcohol, and the tert-butoxycarbonyl group can be removed by reacting the compound in hydrogen fluoride for 30 minutes or by reacting the compound with p-toluene sulfonic acid for 90 minutes.
The compounds of the present invention can be produced in free form or acid salt form according to the method for production. The free form can be easily converted to the acid salt thereof and an acid salt can be easily converted to the free form thereof.
Examples of the acid salts are salts of inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid and phosphoric acid, and salts of organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, citric acid and toluene sulfonic acid.
The chemical structure of the compounds of the present invention was identified by elemental analysis, amino acid analysis, ultraviolet spectrum, and comparison of ultraviolet spectrum of material hydrolyzed with trypsin with 7-amino-4-methylcoumarin.
The compounds of the present invention are all hydrolyzed specifically by one or more enzyme(s) such as Thrombin, Horseshoe Crab Blood Coagulating Enzyme, urokinase and/or Blood Coagulating Factor XII_., and therefore are suitable as synthetic substrates of these compounds.
Amino acids which compose the compounds of the present invention may have any stereochemical configuration, L, D and DL, if the compounds may be enzymatically hydrolyzed.
7-Arginylamino-4-methylcoumarin and the sals thereof are also new compounds. 7-Arginylamino-4-methylcoumarin in the free form can be easily produced by reacting the acid salts thereof with an alkaline material.
The present invention is illustrated in more detail by the following Examples.

Example 1

N"-carbobenzoxy-L-arginine hydrochloric acid salt (69.0 g, 0.2 M) and 7-amino-4-methyl- coumarin (17.5g, 0.1 M) were dissolved in dimethylformamide (DMF) (300 ml). Dicyclohexylcarbodiimide (DCCD) (21 g, 0.1 M) was added to the mixture with stirring at room temperature. This mixture was stirred overnicht at room temperature, and the produced dicyclohexyl urea was removed by filtration. The obtained filtrate was concentrated under reduced pressure and to the obtained residue methyl alcohol (50 ml) and ethyl acetate (500 ml) were added. The precipitated material was separated by filtration to give crude crystals (19 g). These crystals were dissolved in a mixture of heated DMF (30 ml) and heated methyl alcohol (100 ml) and insoluble material was removed by filtration. To the filtrate ethyl acetate (400 ml) was added and the thereby precipitated white crystals were separated by filtration and dried to give 7-(N"-carbobenzoxy-L-arginyl)amino-4-methylcoumarin hydrochloric acid salt (14.5 g, Yield: 29%).

m.p. 210-211 °C (dec.).
[α]¹⁵ _D -17.0° (C = 2.35, DMF).

Elemental Analysis:
7-(N^*-carbobenzoxy-L-arginyl)amino-4-methyl-coumarin hydrochloric acid salt (251 mg, 0.5 mM) was dissolved in a mixture of methyl alcohol (50 ml), acetic acid (5 ml) and water (10 ml), and 5% palladium-carbon catalyst (25 mg) was added thereto. This mixture was stirred under treating with hydrogen gas at room temperature. The catalyst was removed by filtration, and the filtrate was concentrated under reduced pressure. To the residue ether (50 ml) was added and the precipitated powder was separated by filtration to give 7-L-arginyl amino-4-methylcoumarin hydrochloric acid salt 1/2 H₂0 (188 mg, yield: 100%).

m.p. 275°C (dec.)
[α]¹⁵ _D + 91.9° (C = 1.01,25% CH3COOH)

This product gave a single spot on the tin layer chromatogram (silica gel) with a ternary eluent of n-butyl alcohol, acetic acid and water in 4:1:1 volume ratio,
Elemental Analysis:
7-L-arginylamino-4-methylcoumarin hydrochloric acid salt 1/2 H₂0 (1.51 g, 4mM) was dissolved in a mixture of DMF (10 ml) and water (10 ml) and N-hydroxysuccinimide ester of carbobenzoxyglycine (1.53 g, 5 mMO) was added thereto. The mixture was stirred for 15 hours and then concentrated to yield a solid material. This material was purified by column chromatography (2 x 20 cm) using silica gel as adsorbent and a ternary eluent of chloroform, methyl alcohol and acetic acid in 95:5:3 volume ratio. The main fraction was concentrated to yield a solid material and this material was dissolved in acetic acid (50 ml). This solution was lyophilized to give 7-(N"_-carbobenzoxyglycyl-L-arginyl)amino-4-methyl- coumarin hydrochloric acid salt [I] (1.67 g).

m.p. 150°C (dec.)
[α]¹⁵ _D -37.6° (C = 0.56, DMF)

Elemental Analysis:

Example 2

7-(N^α-carbobenzoxyglycyl-L-arginyl)amino-4-methylcoumarin hydrochloric acid salt (559 mg, 1 mM) was dissolved in methyl alcohol (50 ml) and palladium-carbon catalyst (50 mg) was added thereto. This mixture was stirred under treatment with hydrogen gas for 3 hours. The catalyst was removed by filtration and the thus obtained filtrate was concentrated to yield solid material. To the residue ether (50 ml) was added to yield a powder. The powder was separated by filtration and dried to give 7-(glycyl-L-arginyl)amino-4-methylcoumarin hydrochloric acid salt [II] (400 mg).

m.p. 145°C (dec.)
[α]¹⁵ _D -28.5° (C = 0.43, DMF)

Elemental Analysis:

Example 3

7-(N"-glycyl-L-arginyl)amino-4-methylcoumarin hydrochloric acid salt (425 mg, 1 mM) was dissolved in DMF (10 ml), and N-hydroxysuccinimide ester of carbobenzoxy-L-proline (450 mg, 1.3 mM) was added thereto. The mixture was stirred for 15 hours at room temperature and then was concentrated to yield a solid material. This material was purified by column chromatography (2 x 10 cm) using silicagel as an adsorbent and ternary eluent of chloroform, methyl alcohol and acetic acid in 95:5:3 volume ratio. The main fraction was concentrated-to yield a solid material. This material was dissolved in acetic acid (20 ml) and lyophilized to give 7-(N^α-carbobenzoxy-L-prolyl-glycyl-L-arginyl)-amino-4-methylcoumarin hydrochloric acid as the acetic acid adduct [III] (460 mg).

m.p. 200°C (dec.)
[_α]p¹⁵ _D -47.6° (C=0.66, DMF)

Elemental Analysis:

Example 4

7-(N"-carbobenzoxy-L-prolyl-glycyl-L-arginyl)amino-4-methylcoumarin hydrochloric acid salt (200 mg, 0.27 mM) was dissolved in methyl alcohol (50 ml) and 1 N-HCI (0.27 ml) and palladium-carbon catalyst (20 mg) were added thereto. This mixture was stirred under treatment with hydrogen gas for 3 hours. The catalyst was removed by filtration and the filtrate was concentrated to yield a solid material. To the residue ether (50 ml) was added and the thus produced powder was separated by filtration to give 7-(L-prolyl-glycyl-L-arginyl)amino-4-methylcoumarin hydrochloric acid salt [IV] (150 mg).

m.p. 185°C (dec.)
[α]¹⁵ _D -47.7° (C = 1.73, 80% DMF)

Elemental Analysis:

Example 5

N°-carbobenzoxy-N^G-tosyl-L-arginine (See J. A.C.S. 82, 4576) (6.0 g, 13 mM) was dissolved in DMF (20 ml), and 4-methyl-7-aminocoumarin (1.75 g, 10 mM) was added thereto. To this mixture DCCD (2.47 g, 12 mM) was added with ice-cooling. The mixture was stirred overnight at room temperature and the precipitated dicyclohexyl urea was removed by filtration. DMF was distilled off under reduced pressure, and to the residue chloroform (200 ml) was added for extraction. The chloroform solution was washed with 1 N-HCI, 5% NaHC0₃ and water successively, and then was dried over anhydrous sodium sulfate. Next, chloroform was distilled off under reduced pressure, and the residue was purified by column chromatography using silicagel and a binary mixture of chloroform and ethyl acetate in 2:1 volume ratio. The main fractions were combined and concentrated. The residue was dissolved in chloroform (10 ml) and ether was added thereto to precipitate desired material. This material was separated to give 7-(N^α-carbobenioxy-N^G-tosyl-L-arginyl)amino-4-methylcoumarin (2.31 g, yield: 37%).

m.p. 155.0-162.5°C (dec.)
[α]¹⁸ _D +11.2_° (C = 0.5, _DMF)

This product gave a single spot (Rf = 0.4) on the thin layer chromatogram (silicagel) with a ternary eluent of chloroform, methyl alcohol and acetic acid in 95:5:3 volume ratio.
Elemental Analysis:
7-(N^α-carbobenzoxy-N^G-tosyl-L-arginyl)amino-4-methylcoumarin (500 mg) was dissolved in methyl alcohol (50 ml), and 5% palladium-carbon catalyst (50 ml) was added thereto. The mixture was treated with hydrogen gas for 5 hours with stirring. From the reaction mixture, the catalyst was removed by filtration and from the filtrate methyl alcohol was distilled off under reduced pressure. The residue was dissolved in methanol (5 ml) and ether was added to yield a precipitate. The precipitate was separated and dried to give 7-(N^G-tosyl-L-arginyl)amino-4-methylcoumarin (340 mg, yield: 87.1%).

m.p. 174.5-182.5°C (dec.)
[α]¹⁸ _D ―11.4° (C = 0.5, _DMF)

This product gave a single spot (RF = 0.6) on the thin layer chromatogram (silica gel) with a ternary eluent of n-butylalcohol, acetic acid and water in 4:1:1 volume ratio.
Elemental Analysis:
7-(N^G-tosyl-L-arginyl)amino-4-methylcoumarin (490 mg, 1 mM) was dissolved in DMF (5 ml), and N-hydroxysuccinimide ester of carbobenzoxyglycine (459 mg, 1.5 mM) was added thereto. This mixture was stirred for 2 days at room temperature. To this mixture, excess ether was added to yield a precipitate. This precipitate was separated by filtration, dried, and purified by column chromatography (2 x 10 cm) using silica gel as an adsorbent and a ternary eluent of chloroform, ethyl acetate and ethyl alcohol in 5:5:2 volume ratio. The main fractions were combined and concentrated to yield a solid material. The residue was recrystalized from a binary mixture of methyl alcohol (5 ml) and ether (30 ml) to give 7-N^α-carbobenzoxyglycyl-L-N^G-tosylarginyl)amino-4-methylcoumarin (611 mg, yield: 90.1%)

m.p. 130.5―135.5°C
[α]¹⁵ _D -30.1 ° (C = 1, DMF)

Elemental Analysis:

Example 6

7-(N^α-carbobenzoxyglycyl-N^G-tosyl-arginyl)amino-4-methylcoumarin (678 mg, 1 mM) was dissolved in a mixture of methyl alcohol (50 ml) and acetic acid (10 ml), and 5% palladium-carbon catalyst (50 mg) was added thereto. This mixture was treated with hydrogen gas for 4 hours. The catalyst was removed by filtration and from the filtrate, solvent was distilled off under reduced pressure. The residue was dissolved in methyl alcohol (10 ml) and excess ether was added thereto to yield a precipitate.
The precipitate was separated by filtration and dried. The precipitate was dissolved in DMF (8 ml), and N-hydroxysuccinimide ester of tert-butyloxycarbonyl-γ-benzy)-L-glutamic acid 1478 mg. 1.1 mM).. This mixture was stirred for 2 days at room temperature, and excess ether was added to yield a precipitate. This precipitate was separated by filtration and purified by column chromatography (2 x 10 cm) using silica gel as adsorbent and a ternary eluent of chloroform, ethyl acetate and ethyl alcohol in 5:5:2 volume ratio. The main fractions were combined and concentrated to yield a solid material. This material was reprecipitated from a binary mixture of methyl alcohol (10 ml) and ether (50 ml) to give 7-(N^α-tert-butyloxycarbonyl-γ-benzyl-L-glutamylglycyl-N^G-tosyl-L-arginyl)amino-4-methylcoumarin (705 mg, yield 82.0%).

m.p. 118.5-123.5⁰C
[α]¹⁵ _D -12.7° (C = 1, DMF)

Elemental Analysis:

Example 7

To a mixture of 7-(N^α-tert-butyloxycarbonyl-γ-benzyl-L-glutamylglycyl-N^G-tosyl-L-arginyl)amino-4-methylcoumarin (432 mg, 0.5 mM) and anisole (0.5 ml), anhydrous hydrogen fluoride (10 ml) was added thereto at -70°C. This mixture was stirred for 60 minutes under cooling in an ice bath. The excess hydrogen fluoride was distilled off under reduced pressure. To the residue water (5 ml) was added and thus obtained water layer was washed with ether (20 ml) and then passed through a column filled with a ion-exchange resin, Dowex 1 x 2 (acetic acid salt form, 5 ml), which was eluted with water (10 ml). The eluate was lyophilized to give 7-(L-glutamylglycyl-L-arginyl)amino-4-methylcoumarin [V] (225 mg, yield: 74%), which is amorphous.

[α]¹⁵ _D +29.5° (C = 0.5, DMF)

Elemental Analysis:

Example 8

7-(L-glutamylglycyl-L-arginyl)amino-4-methyl-coumarin (100 mg, 0.18 mM) was dissolved in DMF (2 ml), and triethylamine (0.03 ml) was added thereto under stirring and cooling. This mixture was adjusted to pH about 7, and then p-nitrophenyl acetate (36.1 mg, 0.23 mM) was added thereto. This mixture was stirred for 2 days at room temperature. Excess ether was added to the mixture and the formed precipitate was separated by filtration and dried. This product was dissolved in 80% methyl alcohol (2 ml) and ethyl acetate (30 ml) was added thereto for re-precipitation to give 7-(N^α-acetyl-L-glutamylglycyl-L-arginyl)amino-4-methylcoumarin [VI] (99 mg, yield: 84.9%).

m.p. 214.0―217.5°C (dec.)
[α]²⁰ _D -13.8° (_C = ₁, _DMF)

Elemental Analysis:

Example 9

7-(L-glutamylglycyl-L-arginyl)amino-4-methyl-coumarin (100 mg, 0.18 mM) was dissolved in DMF (2 ml), and triethylamine (0.03 ml) was added thereto with stirring and cooling. This mixture was adjusted to pH about 7, and N-hydroxysuccinimide ester of tert-butyloxycarbonyl-L-isoleucine (75.4 mg, 0.23 mM) was added thereto. This mixture was stirred for 3 days at room temperature. To this mixture excess ether was added and the produced precipitate was separated by filtration and dried. This material was dissolved in methyl alcohol (2 ml), and ether (20 ml) was added thereto for reprecipitation to give 7-(N^α-tert-butyloxy-carbonyl-L-isoleucyl-L-glutamylglycyl-L-arginyl)amino-4-methylcoumarin [VII] (120 mg, yield: 86.3%).

m.p. 208.0-212.0°C (dec.)
[α]²⁰ _D -16.7° (C = 1, DMF)

Elemental Analysis:

Example 10

7-(N"-glycyl-L-arginyl)amino-4-methylcoumarin hydrochloric acid salt (255 mg, 0.6 mM) was dissolved in a mixture of DMF (1 ml) and water (1 ml), and glutaric acid anhydride (75.2 mg, 0.66 mM) was added thereto. This mixture was stirred for 15 hours at room temperature, and ether (50 ml) was added to yield a precipitate. The precipitate was separated by filtration, dried and purified by column chromatography (2 x 15 cm) using silica gel as adsorbent and a binary eluent of chloroform and methyl alcohol in a 1:2 volume ratio. The main fraction was concentrated to yield a solid material. This material was re-recipitated from DMF (5 ml)-ether (5 ml) to give 7-(N^α-glutaryl-glycyl-L-arginyl)amino-4-methylcoumarin hydrochloric acid salt [VIII] (250 mg, yield: 77.4%).

m.p. 234.5―238.0°C (dec.)
[α]¹⁵ _D + 23.3° (C = , DMF)

Elemental Analysis:

Example 11 -

7-L-arginylamino-4-methylcoumarin hydrochloric acid salt 1/2 H₂0 (377 mg, 1 mM) was dissolved in a mixture of DMF (10 ml) and water (10 ml), and N^α-tert-butyloxycarbonyl-L-valyl-L-seryl- glycine-N-hydroxysuccinimide ester (458 mg, 1 mM) was added thereto. This mixture was stirred for 15 hours at room temperature and concentrated to yield a solid material. The residue was purified by column chromatography (2 x 15 cm) using silicagel as adsorbent and ternary mixtures of chloroform, methyl alcohol and acetic acid in 95:5:3 (adsorption) and then 85:20:5 (elution) in volume ratio. The main fraction was concentrated to yield a solid material. The material was dissolved in acetic acid (50 ml) and lyophilized to give 7-(N^α-tert-butyloxycarbonyl-L-valyl-L-seryl-glycyl-L-arginyl)amino-4-methyl- coumarin hydrochloric acid salt [IX] (320 mg).

m.p. 160°C (dec.)
[α]²¹ _D-21.1°(C=1.15,80%DMF)

Elemental Analaysis:

Example 12

7-(N^α-tert-butyloxycarbonyl-O-benzyl-L-serylglycyl-L-arginyl)amino-4-methylcoumarin hydrochloric acid salt (120 mg, 0.28 mM) was dissolved in methyl alcohol (50 ml), and palladium-carbon catalyst (20 mg) was added. Through this mixture hydrogen gas was passed for 4 hours with stirring. The catalyst was removed by filtration and the filtrate was concentrated to yield a solid material. The residue was dissolved in acetic acid (10 ml) and lyophilized to give 7-(N"-tert-butyloxycarbonyl-L-seryl- glycyl-L-arginyl)amino-4-methylcoumarin hydrochloric acid salt [X] (70 mg).

m.p. 180°C (dec.)
[α]²¹ _D -28.4° (C = 0.44, 80% DMF)

Elemental Analysis:

Example 13

7-(N"-glycyl-L-arginyl)amino-4-methyl-coumarin hydrochloric acid salt (230 mg, 0.54 mM) was dissolved in DMF (5 ml), and N^α-tert-butyloxycarbonyl-O-benzyl-L-serine-N-hydroxysuccinimide ester (253 mg, 0.65 mM) was added thereto. This mixture was stirred for 15 hours at room temperature and concentrated to yield a solid material. The material was purified by column chromatography (2 x 15 cm) using silica gel as adsorbent and ternary mixtures of chloroform, methylalcohol and acetic acid in 95:5:3 (adsorption) and then 85:20:5 (elution) in volume ratio. The main fraction was concentrated to yield a solid material and dissolved in acetic acid (20 ml). This solution was lyophilized to give 7-(N"- tert-butyloxycarbonyl-O-benzyl-L-serylglycyl-L-arginyl)-amino-4-methyl-coumarin hydrochloric acid salt [Xi] (180 mg).

m.p. 160°C (dec.)
[α]²¹ _D -29.6_° (_C = ₀.₄₆, _{80% DMF})

Elemental Analysis:

Example 14

7-L-arginylamino-4-methylcoumarin hydrochloric acid salt 1/2 H₂O (377 mg, 1 mM) was dissolved in a mixture of DMF (5 ml) and water (5 ml), N°-tert-butyloxycarbonyl-L-valyl-L-leucyl- glycine-N-hydroxysuccinimide ester (484 mg, 1 mM) was added thereto. This mixture was stirred for 15 hours at room temperature and concentrated to yield a solid material. The material was purified by column chromatography (2 x 15 cm) using silica gel as adsorbent and ternary mixtures of chloroform, methylalcohol and acetic acid in 95:5:3 (adsorption) and then 85:20:5 (elution) in volume ratio. The main fraction was concentrated to yield a solid material, dissolved in acetic acid (20 ml) and lyophilized to give 7-(N°-tert-butyloxycarbonyl-L-valyl-L-leucyl-glycyl-L-arginyl)amino-4-methylcoumarin hydrochloric acid salt [XII] (350 mg).

m.p. 150°C (dec.)
[α]²¹ _D -35.9° (C = 2.16, 80% DMF)

Elemental Analysis:

Example 15

7-(N^*-glycyl-L-arginyl)amino-4-methylcoumarin hydrochloric acid salt (91.4 mg, 0.215 mM) was dissolved in DMF (5 ml), and N-tert-butyloxycarbonyl-L-leucine-N-hydroxysuccinimide ester (85 mg, 0.26 mM) was added thereto. This mixture was stirred for 15 hours at room temperature and concentrated to yield a solid material. The material was purified by column chromatography (1 x 15 cm) using silica gel as adsorbent and ternary mixtures of chloroform, methyl alcohol and acetic acid in 95:5:3 and then 85:20:5 volume ratio. The main fraction was concentrated to a solid material, then dissolved in acetic acid (20 ml) and lyophilized to give 7-(N"-tert-butyloxycarbonyl-L-leucylglycyl-L-arginyl)amino-4-methylcoumarin hydrochloric acid salt [XIII] (120 mg).

m.p. 160°C (dec.)
[α]²¹ _D -37._8° (_C = ₀.₆₃, _{80% DMF})

Elemental Analysis:

Example 16

7-(N"-glycyl-L-arginyl)amino-4-methylcoumarin hydrochloric acid salt (136 mg, 0.32 mM) was dissolved in DMF (5 ml), and Nα-carbobenzoxy-L-leucine-N-hydroxysuccinimide ester (140 mg, 0.38 mM) was added thereto. This mixture was stirred for 15 hours at room temperature and concentrated to yield a solid material. The material was purified by column chromatography (1 x 15 cm) using silica gel and ternary mixtures of chloroform, methyl alcohol and acetic acid in 95:5:3 and then 85:20:5 volume ratio. The main fraction was concentrated to yield a solid material, dissolved in acetic acid (20 ml) and lyophilized to give 7-(N"carbobenzoxy-L-leucyl-glycyl-L-arginyl)amino-4-methylcoumarin hydrochloric acid salt [XIV] (150 mg).

m.p. 150°C (dec.)
[α]²¹ _D -35.1 °(C = 1.21, 80% DMF)

Elemental Analysis:

Example 17

7-L-arginylamino-4-methylcoumarin hydrochloric acid salt 1/2 H₂O (754 mg, 2 mM) was dissolved in a mixture of DMF (10 ml) and water (10 ml), and carbobenzoxy-L-phenylalanine-N-hydroxysuccinimide ester (1.03 g, 2.6 mM) was added thereto. This mixture was stirred for 15 hours at room temperature and concentrated to yield a solid material. The material was purified by column chromatography (2 x 15 cm) using silica gel as adsorbent and a ternary mixture of chloroform, methylalcohol and acetic acid in 95:5:3 volume ratio. The main fraction was concentrated to yield a solid material, dissolved in acetic acid (30 ml) and lyophilized to give 7-(N"-carbobenzoxy-L-phenylalanyl-L-arginyl)amino-4-methylcoumarin hydrochloric acid salt [XV] (920 mg).

m.p. 150°C (dec.)
[α]¹⁵ _D -23.4° (C = 0.65, DMF)

Elemental Analysis:

Example 18

7-(N^a-carbobenzoxy-L-phenylalanyl-L-arginyl)amino-4-methylcoumarin hydrochloric acid salt (1.3 g, 2 mM) was dissolved in methyl alcohol (100 ml), and palladium-carbon catalyst (100 mg) was added. Through this mixture hydrogen gas was passed for 3 hours with stirring. The catalyst was removed by filtration and the filtrate was concentrated to yield a solid material. To the material ether (100 ml) was added and this produced powder was separated by filtration to give 7-(L-phenylalanyl-L-arginyl)amino-4-methylcoumarin hydrochloric acid salt [XVI] (1.0 g).

m.p. 140°C (dec.)
[α]¹⁵ D-33.6° (C = 0.52, DMF)

Elemental Analysis:

Example 19

7-(L-phenylalanyl-L-arginyl)amino-4-methylcoumarin hydrochloric acid salt (515 mg, 1 mM) was dissolved in DMF (5 ml), and carbobenzoxy-L-proline-N-hydroxysuccinimide ester (450 mg, 1.3 mM) was added thereto. This mixture was stirred for 15 hours and concentrated to yield a solid material. The material was purified by column chromatography (2 x 15 cm) using silica gel as an adsorbent and a ternary mixture of chloroform, methyl alcohol and acetic acid in 95:5:3 volume ratio. The main fraction was concentrated to yield a solid material. The solid material was dissolved in acetic acid and lyophilized to give 7-(N'-carbobenzoxy-L-prolyl-L-phenylalanyl-L-arginyl)amino-4-methylcoumarin hydrochloric acid salt as adduct of acetic acid [XVII] (510 mg).

m.p. 160°C (dec.)
[α]¹⁵ _D -₅₃._6° (C = 0.75, _DMF)

Elemental Analysis:

Example 20

7-(N^α-carbobenzoxy-L-prolyl-L-phenylalanyl-L-arginyl)amino-4-methylcoumarin hydrochloric acid salt (200 mg, 0.27 mM) was dissolved in methyl alcohol (50 ml), and 1 N-HCI (0.27 ml) and palladium-carbon catalyst (20 mg) were added thereto. Through this mixture hydrogen gas was passed with stirring for 3 hours. The catalyst was removed by filtration and the filtrate was concentrated to yield a solid residue. To the residue ether (50 ml) was added and this produced powder was separated by filtration to give 7-(L-prolyl-L-phenylalanyl-L-arginyl)amino-4-methylcoumarin hydrochloric acid salt [XVIII] (150 mg).

m.p. 185°C (dec.)
[α]²¹ _D -3₇._5° (_{C = 1}.₅₂, _{80% DMF})

Elemental Analysis:

Example 21

7-L-arginylamino-4-methylcoumarin hydrochloric acid salt 1/2 H₂0 (377 mg, 1 mM) was dissolved in a mixture of DMF (10 ml) and water (10 ml), and carbobenzoxy-L-proline-N-hydroxysuccinimide ester (450 mg, 1.3 mM) was added thereto. This mixture was stirred for 15 hours at room temperature and concentrated to yield a solid material. The solid material was purified by column chromatography (2 x 15 cm) using silica gel as adsorbent and ternary mixtures of chloroform, methyl alcohol and acetic acid in 95:5:3 and then 85:20:5 volume ratio. The main fraction was concentrated to yield a solid material, dissolved in acetic acid (10 ml), and lyophilized to give 7-(N"-carbobenzoxy-L-prolyl-L-arginyl)amino-4-methylcoumarin hydrochloric acid salt [XIX] (530 mg).

m.p. 198°C (dec.)
[α]¹⁵ _D -77.0° (C = 0.6, DMF)

Elemental Analysis:
The carbobenzoxy group of the compound thus obtained can be removed by applying a hydrogenation reaction in alcohol thereto.

Example 22

7-L-arginylamino-4-methylcoumarin hydrochloric acid salt 1/2 H₂0 (377 mg, 1 mM) was dissolved in a mixture of DMF (5 ml) and water (5 ml), and tert-butyloxycarbonyl-L-valyl-L-proline-p-nitrophenyl ester (566 mg, 1.3 mM) was added thereto. The mixture was stirred for 15 hours at room temperature and concentrated to yield a solid material. The material was purified by column chromatography (2 x 15 cm) using silica gel as adsorbent and ternary mixtures of chloroform, methyl alcohol and acetic acid in 95:5:3 and then 85:20:5 volume ratio. The main fraction was concentrated to yield a solid material, dissolved in acetic acid (10 ml) and lyophilized to give 7-(N"-tert-butyloxycarbonyl-L-valyl-L-prolyl-L-arginyl)amino-4-methylcoumarin hydrochloric acid salt [XX] (320 mg).

m.p. 155°C (dec.)
[α]¹⁵ _D -64.8° (C = 0.66, DMF)

Elemental Analysis:
The compound produced above (0.1-0.2 mM) was dissolved in a mixture of dimethylsulfoxide (5 ml) and water (5 ml) and this solution was diluted with 0.05 M Tris-HCI buffer (pH 8.0) containing both 0.1 M NaCl and 10 mM CaCI₂ to adjust the total volume thereof to 500 ml and thereby a substrate solution was prepared.
Each substrate solution (2 ml) was added in a test tube and the obtained test tube was kept at 37°C for 5 minutes. Then, each enzyme solution (20 µl) was added thereto and incubated at 37°C for 20 minutes with stirring. The incubation was stopped by adding 100% acetic acid (0.5 ml) thereto.
The hydrolysis degree was determined by measuring the fluorescence intensity at 460 nm with excitation at 380 mn in the fluorescence spectra. Results are listed in the following table.
Each value is the fluorescence intensity ratio when the fluorescence intensity of 7-amino-4-methylcoumarin solution (40 µM, 40 µmol/l) being 100.
A value having the mark -- ^*-- is one with the fluorescence intensity of 7-amino-4-methylcoumarin solution (200 µM, 200 µmol/l) being 100.
In these experiments each enzyme was dissolved in the above buffer solution and used.

Claims

1. 7-(N^a-R-X-arginyl)-amino-4-methylcoumarins of the general formula

and acid salts thereof,

wherein X is a residue of an amino acid selected from the group of glycine, phenylalanine and proline, and, when

a) X is the residue of glycine, R is selected from a hydrogen atom and prolyl, glutamyl, seryl, leucyl, isoleucyl-glutamyl, valylseryl and valylleucyl groups,

b) X is the residue of phenylalanine, R is selected from the group consisting of a hydrogen atom and a prolyl group and

c) X is the residue of proline, R is selected from the group consisting of a hydrogen atom and a valyl group,

and wherein N^α-amino and imino groups, hydroxy groups, carboxyl groups and guanidino groups of the compounds may be protected by conventional protecting groups for peptides.