CS262120B1 - Chronogenic substrates for priitealytic enzymes cleaving in the acidic pH range - Google Patents

Abstract

The present invention relates to chromogenic substrates for proteolytic enzymes that cleave in the acidic pH range. It is a group of novel compounds of the type of acylated tetrapeptide derivatives of p-nitroanilide useful in determining the enzyme activity of aspartic proteinases.

Description

The invention relates to chromogenic substrates for proteolytic enzymes cleaving in the acidic pH range of the general formula ^N ° 2 in which:

X is hydrogen, C 3 -C 5 -carboxyalkanecarbonyl or C 1 -C 5 alkanecarbonyl,

And the remainder pyroglutamic acid, aspartic acid, glutamic acid, glycine, leucine or 2-oxoimidazolidine-1-carbonyl,

B a histidine, glycine, serine or proline residue,

C is a phenylalanine, leucine, norleucine, methionine or S-alkylcysteine residue, wherein the alkyl has 1 to 3 carbon atoms.

The compounds of the above formula, the nature of tri- and tetrapeptides, are chromogenic substrates for the determination of proteolytic enzymes cleaving in the acidic pH range.

Compared to similar, hitherto known and used chromogenic substrates [Bioorg. Khimija 9, 470 (1983); 10, 390 (1984)], the substrates of the formula exhibit high water solubility and high assay sensitivity.

Chromogenic substrates of the formula may be prepared by methods commonly used in peptide synthesis, for example by the azide, anhydride, carbodiimide or active ester method.

Notes on the experimental part: Chromatography was performed on a thin layer of silica gel (Kiesel-gel, Merck) in the following systems:

System S _t :

1-butanol - acetic acid - water (4:

: 1: 1);

System S ₂ :

1-butanol-acetic acid-pyridine-water (15: 3: 10: 6).

The standard method is to treat the substance by dissolving it in ethyl acetate and gradually shaking it with 1M hydrochloric acid, water, 5% sodium bicarbonate, water, drying over anhydrous sodium sulfate and evaporating in vacuo.

Example 1 t-butyloxycarbonylphenyl alanyl-phenylalanine p-nitroanilide

To a solution of tert-butyloxycarbonylphenylalanine (15.9 g; 60 mmol) and N-hydroxybenztriazole (6.9 g; 60 mmol) in dimethylformamide (100 mL) cooled to -5 ° C was added N, N'-dicyclohexylcarbodiimide (13, 2 g). After stirring and cooling at 0 degrees C for 1 h, a solution of phenylalanine p-nitroanilide (17.1 g; 60 mmol) in dimethylformamide (30 mL) was added. After stirring at 0 ° C for 1 h and standing at room temperature for 12 h, the precipitated N, N'-dicyclohexylurea was filtered off, washed with tetrahydrofuran and the filtrate was evaporated. The crystalline residue was worked up in a standard manner. Crystallization from ethylnatébo acetate (200 mL) and petroleum ether (800 mL) afforded 26.5 g (83%) of product, mp 179-180 ° C, [jo ^{1 20} + 15.8 ° (C - 0.2; methanol). phenylalanyl-phenylalanine p-nitroanilide

To a solution of p-nitroanilide tert-butyloxycarbonylphenylalanyl-phenylalanine (21.3 g; 40 mmol) in ice. a solution of 2M hydrochloric acid in ice was added to acetic acid. acetic acid (60 mL). After standing at room temperature for 3 h, the crystalline hydrochloride was precipitated by the addition of ether (1550 ml), filtered and dried in a desiccator over phosphorus pentoxide. Then the hydrochloride was dissolved in 50% ethanol (aq) (150 ml) and the pH of the solution was adjusted to 7-8 by the addition of concentrated ammonia.

8. The precipitated crystalline base was immediately filtered off, washed with cold water, air-dried and dried over phosphorus pentoxide. 12.4 g (72%) of the product with m.p. 168-169 ° C were obtained; [α] D ²⁰ + 42.2 ° (c ~ 0.2; methanol).

p-Nitroanilide pyramlutamyl-histidyl-phenylalanylphenylalanine (acetate)

To a solution of pyroglutamyl-histidine hydrazide (2.8 g; 10 mmol) in dimethylformamide (100 mL) and concentrated hydrochloric acid (4 mL) cooled to -20 degrees Celsius was added with stirring a solution of sodium nitrite (0.7 g) in water (2.8 mL). After 10 min. with stirring and cooling (1010 ° C), the pH of the reaction solution was adjusted to 6.9-7.1 with N-ethylpiperidine, and a solution of phenylalanyl-phenylalanine p-nitroanilide (4.32 g; 10 mmol) in dimethylformamide (100 mL) pre-cooled at -10 ° C. After standing at +3 ° C for 12 h, the reaction solution was evaporated, the residue was stirred with water and after 2 h the product was filtered and crystallized from e8.

282120

With thanol (50 ml) and ether (250 ml). Recrystallization from acetic acid (30 mL) and ethyl acetate (100 mL) afforded 3.1 g (42%] product, mp 238-240 ° C; [ «Id -1-10,9 ²⁰ (c - 0.2 dimethylformamide).

Example 2 p-Nitroanilide tert-butyloxycarbonylglycyl-glycylphenylalanyl-phenylalanine

To a solution of tert-butyloxycarbonylglycyl-glycine (1.67 g; 5 mmol) and phenylalanyl-phenylalanine p-nitroanilide (2.16 g; 5 mmol) in dimethylformamide (70 mL) cooled to 0 ° C was added N, N'- dicyclohexylcarboidiimide (1.1 g). After stirring for 2 h and standing at room temperature for 12 h, the precipitated N, N'-dicyclohexylurea was filtered off, washed with dimethylformamide; the filtrate was evaporated and the residue was worked up in a standard manner. The crystalline residue was recrystallized from 2-propanol (50 ml) and petroleum ether (300 ml). 2.1 (65%) of m.p. 197-199 ° C; [.alpha.] @ ²⁰ +16.7 DEG (c = 0.2; dimethylformamide).

Glycyl-glycyl-phenylalanyl-phenylalanine p-nitroanilide hydrochloride

To a solution of t-butyloxycarbonylglycyl-glycyl-phenylalanyl-alanine p-nitroanilide (1.94 g; 3 mmol) in ice. acetic acid (9 mL) was added a solution of 2M hydrochloric acid in ice. acetic acid (6 mL). After standing at room temperature for 3 h, the crystalline hydrochloride was filtered off, the proonyl ether and dried over sodium hydroxide and phosphorus pentoxide. 1.8 g (quant.) Of the product were obtained, mp 232-236 ° C. The sample to be analyzed was crystallized from acetic acid and ether; mp 254-257 ° C, [α] ²⁰ D + 2.7 ° (c - 0.2; dimethylformamide).

glycyl-glycyl-phenylalanyl-phenylalanine p-nitroanilide

A solution of glycyl-glycyl-phenylalanyl-phenylalanine p-nitroanilide hydrochloride (1.9 g) in methanol (50 ml) and water (25 ml) was adjusted to pH 7-8 with 10% ammonia, diluted with water (75 ml) and precipitated. the precipitate was filtered off, washed with water, air dried and dried over phosphorus pentoxide; 1.3 g (72%) of m.p. 185-188 ° C were obtained.

p-Nitroanilide glutaryl-glycyl-glycyl-phenylalanyl-phenylalanine

To a solution of glycyl-glycyl-phenylalanyl-phenylalanine p-nitroanilide (550 mg; 1 mmol) in dimethylformamide (10 mL) was added glutaric anhydride (230 mg) and the solution was heated at 80 ° C for 1 h, then evaporated and stirred. with water (20 ml) and the resulting precipitate was filtered off and washed with chilled water; the crude product was recrystallized from dilute acetic acid. 530 mg (79%) of the product with mp 221-224 C, [α] _D ²⁰ + 13.4 ° (c = 0.2, dimethylformamide), was obtained.

Example 3 t-Butyloxycarbonylphenylalanyl-methionine p-nitroanilide

Prepared in a similar manner to the corresponding dipeptide p-nitroanilide tert-butyloxycarbonylphenylalanyl-phenylalanine in Example 1 in a yield of 68%, m.p. 199-200 ° C.

Phenylalanylmethionine p-nitroanilide hydrochloride

Prepared in a similar manner to the corresponding phenylalanyl-phenylalanine p-nitroanilide dipeptide shown in Example 1 in a yield of 92%; the sample to be analyzed was crystallized from methanol and ether, mp 246-248 ° C. [ατ] ο ^{2 (} + + 87.7 ° (c = 0.2; methanol).

Pyroglutamyl-histidyl-phenylalanylmethtonine p-nitroanilide

Prepared in a similar manner to the corresponding tetrapeptide of Example 1, from pyroglutamylhistidine hydrazide and phenylalanyl-methionine p-nitroanilide in a yield of 46%, mp 228-231 ° C.

Example 4 2-Oxoimidazolyl-1-carbonylhistidylphenylalanyl-phenylalanine p-nitroanilide

It was prepared in a similar manner to the corresponding pyroglutamyl derivative shown in Example 1, from 2-oxoimidazolidine-1-carbonyl-histidine hydrazide and phenylalanyl-phenylalanine p-nitroanilide in a yield of 46 why. mp 169-172 ° C; [.alpha.] D @ ²⁰ -8.9 DEG (c = 0.2; methanol).

Example 5 Acetylhistidine-phenylalanyl p-nitroanilide. -phenylalanine

It was prepared in a similar manner to the tetrapeptide shown in Example 1, i.e. by the azide method from acetylhistidine hydrazide (Collection Czechoslov. Chera. Commun. 45, 442 (1980)] of phenylalanyl-phenylalanine p-nitroanilide in 52% yield, mp 138-142 degrees Celsius. .

Example 6 t-Butyloxycarbonylphenylalanyl-S-methylcysteine p-nitroanilide

Prepared in a similar manner to the corresponding dipeptide of Example 1, from tert-butyloxycarbonylphenylalanine and S-methylcysteine p-nitroanilide in 77% yield, mp 132-135 ° C.

Phenylalanyl-S-methylcysteine p-nitroanilide hydrochloride

It was prepared in a similar manner to the phenylalanyl-methionine p-nitroanilide hydrochloride shown in Example 3 in a yield of 90%, m.p. 178-181 ° C.

Pyroglutamyl-histidyl-phenylalanyl-S-methyl-tysteine p-nitroanilide

It was prepared in a similar manner to the corresponding tetrapeptide shown in Example 1 in a yield of 56%, mp 142-145 ° C.

Example 7

Benzyloxycarbonylleucylserine hydrazide

To a solution of benzyloxycarbonylleucylserine methyl ester (7.35 g) in methanol (50 mL) was added 100% hydrazine hydrate (5 mL) After standing at room temperature for one day, the precipitated hydrazide was filtered off and crystallized from ethanol to give 5.8 g ( 79%) m.p. 175-177 ° C.

Benzyloxycarbonylleucyl-seryl-phenylalanyl-phenylalanine p-nitroanilide

It was prepared in a similar manner to the corresponding tetrapeptide of Example 1, from the corresponding benzyloxycarbonylleucylserine hydrazide and phenylalanyl-phenylalanine p-nitroanilide in a yield of 59%, mp 212-214 ° C.

leucyl-seryl-phenylalanyl-phenyl alanine p-nitroanilide

To a solution of benzyloxycarbonylleucyl-seryl-phenylalanyl-phenylalanine p-nitroanilide (1.6 gj in glacial acetic acid (5 mL) was added 37% hydrogen bromide in glacial acetic acid. After standing at room temperature for 1 h After 12 h, the hydrobromide was dissolved in ethanol (10 mL) and the base was liberated by the addition of 5% sodium bicarbonate (5 mL), after 2 h the base was filtered off, washed with water Crystallization from a mixture of ethyl acetate (100 ml), dimethylformamide (5 ml) and petroleum (150 ml) gave 520 mg (41%) of mp 201-203 ° C (melt at 126 degrees Celsius).

glutaryl-leucyl-seryl-phenylalanyl-phenylalanine p-nitroanilide

To a solution of leucyl-seryl-phenylalanyl-phenylalanine p-nitroanilide (255 mg) in dimethylformamide (3 mL) was added glutaric anhydride (100 mg) and the reaction solution was heated at 80 ° C for 1 h. Then the solution was evaporated, the product precipitated with water, filtered off, washed with water and crystallized from 2-propanol (30 ml) and water (0.1 ml); 190 mg (67%) of m.p. 256 DEG-259 DEG C. was obtained. The sample to be analyzed was crystallized similarly; mp 258-260 ° C. Example 8 t-Butyloxycarbonylphenylalanyl-leucine p-nitroanilide

It was prepared analogously to the corresponding dipeptide given in Example 1, in quantitative yield, m.p. 151-153 ° C.

Phenylalanyl-leucine p-nitroanilide hydrochloride

Prepared in a similar manner to the corresponding phenylalanyl-methionine p-nitroanilide dipeptide shown in Example 3 in a yield of 88%; R _f = 0.77 / _x S; 0.81 / S ₃ .

t-butyloxycarbonylleucyl-seryl-phenylalanyl-leucine p-nitroanilide

It was prepared analogously to the corresponding tetrapeptide of Example 7 in a yield of 55% and mp 203-205 ° C.

leucyl-seryl-phenylalanyl-leucine p-nitroanilide

It was prepared analogously to the corresponding free tetrapeptide shown in Example 7 in a yield of 54% and mp 197-200 ° C. Rf = 0.69 / Si; 0.82 / S ₂ .

glutaryl-leucyl-seryl phenylalanyl-leucine p-nitroanilide

It was prepared analogously to the corresponding glutaryltetrapeptide shown in Example 7 in a yield of 89% and mp 273-275 ° C. Example 9 t-Butyloxycarbonylphenylalanyl-norleucine p-nitroanilide

It was prepared in quantitative yield as the analogous dipeptide shown in Example 1; mp 166-167 ° C.

6 2 1 6, 0

Phenylalanyl-norleucine p-nitroanilide hydrochloride

It was prepared in a similar manner to the corresponding dipeptide of Example 8 in 84% yield; Rf = 0.73 / Si; 0.74 / S ₂ .

Benzyloxycarbonylglycyl • -propyl-phenylanyl-nor leucine p-nitroanilide

To a solution of benzyloxycarbonylglycyl-proline (460 mg) in dimethylformamide (15 mL) and N-hydroxysuccinimide (175 mg) cooled to -20 ° C was added N, N‘-dicyclohexylcarbodiimide (330 mg). After stirring and cooling (-5 ° C) for 1 h, a solution of phenylalanyl-norleucine p-nitroanilide in dimethylformamide (10 mL), released from the corresponding hydrochloride (652 mg), was added to the reaction solution by addition of N-ethylpiperidimine (0.21 mL). . After stirring for 2 h and standing at room temperature for 12 h, the precipitated N, N‘-dicyclohexylurea was filtered off, the filtrate was evaporated and the residue was precipitated with water. Crystallization from ethanol (5 ml) and water (20 ml) gave 1.02 g (99%) of the product, m.p. 101-104 ° C.

glycyl-prolyl-phenylalanyl-norleucine p-nitroanilide

It was prepared in a similar manner to the corresponding tetrapeptide shown in Example 7 in a yield of 64% and mp 12-212 ° C. R _f = 0.69 / 0.83 Sn / S _{2 l.}

glutaryl-glycyl-prolyl-phenylalanyl-norleucine p-nitroanilide

It was prepared in a similar manner to the corresponding glutaryl tetrapeptide shown in Example 7 in a yield of 84% and mp 128-130 ° C.

Claims (1)

Chromogenic substrates for proteolytic enzymes cleaving in the acidic pH range of the general formula λ -A-3-Ph & X is hydrogen, C 3 -C 5 -carboxyalkanecarbonyl or C 1 -C 5 alkanecarbonyl, And the remainder pyroglutamic acid, aspartic acid, glutamic acid, glycine, leucine or 2-oxoimidazolidine-1-carbonyl, B a histidine, glycine, serine or proline residue, C is a phenylalanine, leucine, norleucine, methionine or S-alkylcysteine residue, wherein the alkyl has 1 to 3 carbon atoms.