CS244022B1 - Dipeptide or tr (arylamides and C-terminal S-aralkylcysteine residues and process for their production) - Google Patents

Abstract

Arylamides of dipeptides or tripeptides with a C-terminal S-aralkylcysteine residue of the general formula I X - A - B - Cys/Y/ - NH - Z /1/, where X represents an alkanecarbonyl residue of 62 to C6, -carbosialkanecarbonyl residue of C. al C6, A is a glycine, leucine, renylalanine, methionine residue or zero, B is a glycine, alanine, leucine, phenylalanine or methionine residue, Y is benzyl or p-nitrobenzyl and Z is p-nitrophenyl, are new compounds, useful as chromogenic substrates for determining the activities of proteolytic enzymes, in particular papain, and can be prepared, for example, from a compound of the general formula 11 Cys/Y/ - NH - Z /11/, where Y and Z have the above meaning, by β-condensation protected with a compound W-A or W-A-B, where A and B have the above meaning and W is a protecting group, and then introducing the residue X with the above-mentioned meaning into the resulting intermediate, after removal of the protecting group V.

Description

The invention relates to arylamides of dipeptides or tripeptides having a C-terminal S-argkylcysteine radical of formula 1,

X - A - B - Cys (Y) - NH - Z (I), wherein X represents an alkanercarbonyl shytoc Cg to Cg,

- a carboxyalkanecarbonyl radical C 4 to C 8,

A is a residue of glycine, leucine, phenylalanine, methionine or zero,

B is a residue of glycine, alanine, leucine, phenylalanine or methlonine,

X is benzyl or p-nitrobenzyl; and

Z is p-nitrophenyl.

The compounds of the formula I which are novel are useful as specific chromogenic substrates for proteolytic enzymes, in particular papain. Papain is used in the pharmaceutical industry, eg in the production of dietetic preparations, stabilizers for beer, for the maturation of meat products. Monitoring papain activity, especially in the food industry, is technically important.

The determination of papain activity in beer is still carried out, for example, by immunological methods, probably due to its wide substrate non-specificity. Although a number of papers have been published following the mapping of synthetic substrates for papain, specific substrates have not yet been discovered that would reliably allow the determination of small quantities of this enzyme.

Surprisingly, the inventors have found that low molecular weight peptides with a C-terminal S-aralkylcysteine residue are suitable and very specific substrates for papain allowing reliable determination of 10? up to 10 ~ g / ml of this enzyme. Practically this means that these synthetic substrates are comparable in sensitivity and specificity to the existing conventional immunological method.

Compared to this method, the synthetic substrate method is simpler, less time consuming and thus economically advantageous. The advantages of the new synthetic substrates are evident from Table Γ, which gives the kinetic constants of the hydrolysis. The kinetic data of the synthetic substrates of Formula 1 are better than the kinetic data for papain substrates published, e.g., in Biochem. <J. 219, 325 (1984).

Table 1

Kinetic constants of chromogenic substrates cleaved by papain at 25 ° C

Substrate (miles) ^to cat Ca '') ^to cat ^{/ K} M (M ''. s') Suc-Gly-Cys (Bzl) -NAn 5.6 0.18 32 Suc-Gly-Gly-Oy with (Bz1) -NAn 2.8 0.16 57 Suc-Ala-Cys (Bzl) -NAn 2,0 1,2 600 Suc-Leu-Leu-Cys (Bzl) -NAn 0.11 2.2 20 000 Suc = succinyl; NAn ^x p-nitroanilide Bzl ¹ benzyl Alenine (Ala), Leucine (Leu), cystine (Cys) are L-configurations,

The arylamides of the dipeptides and tripeptides having the C-terminal S-aralkylcysteine radical of formula 1 can be prepared according to the invention in several ways. For example, by first treating a compound of formula 11

Cya (Y) - NH - Z (IX), in which Y and Z are the same as in formula I, condenses with WAB, where A and B are the same as in formula I and W protecting a protected compound WA or a group such as benzyloxy3 carbonyl followed by introducing into the prepared intermediate, after removal of the protecting group V, a radical X, wherein X denotes the same as in formula I, or by introducing a compound of the formula II

Cys (X) -NH-3 (II), wherein X and Z are the same as in Formula I, condenses with the protected compound XA or XAB, wherein X and A and B are the same as in Formula I, to form a compound of Formula I or by first treating a compound of formula II

Cys (X) - NH - Z (II) in which X and Z denote the same as in formula I condenses with a compound of formula III W - Β (III) in which B denotes the same as in formula I and V protecting group, for example a banzylocarbonyl group, whereupon the resulting compound of formula IV is formed

W - B - Cyya (X) - HH - Z (IV), in which B and X and Z denote the same in formulas I and V as in formula III, after deprotection W, condenses with a compound of formula V

W - A (V), in which A denotes the same as in formula I and I the same as in formula III, after removal of the protecting group W a residue X is incorporated into the resulting peptide or so that a compound of formula II

Cys (X) - NH - Z (II), where X and Z are the same as in formula I, is condensed with a compound of formula VI X - Β (VI), or with a compound of formula VII

X-A-B (VII), wherein X and A and B are the same as in I, to form a compound of Formula 1.

The condensation reactions were carried out using the azide method and the carbodiimide method, but other methods used in peptide synthesis may also be used.

Further preparation details are given in the following examples:

Example p-Nitroenilide benzyloxycarbonylalanyl-S-benzylcysteine

To a solution of S-benzylcysteine p-nitroanilide (3.35 g; 10 mmol) and benzyloxycarbenylalanine (2.25 g; 10 mmol) in dimethylformamide (60 mL) cooled to 0 ° C was added Ν, β-dicyclo hexylcarbodiimide (2). , 2 g).

o. After stirring at 0 ° C for 2 h and standing at room temperature for 12 h, the precipitated N, N -dicyclohexylurea was filtered off, washed with dimethylformamide and the filtrate was evaporated in vacuo. The residue was dissolved in ethyl acetate (100 mL) and the solution was shaken successively with 1 H acid. In a line of hydrochloric acid and 5% sodium bicarbonate, water, dried over anhydrous sodium sulfate, and evaporated in vacuo. The solid residue was crystallized from 2-propanol (25 ml) by the addition of petroleum ether. 3.8 g (7)% of the product were obtained. The sample to be analyzed was crystallized similarly, m.p. 156-157 ° C.

p-Nitroanilld 3-carbexypropionylalanyl-S-benzylcyatin

To a solution of benzyloxycarbonylalanyl-S-benzylcyatinine p-nitroanilide (1.6 g; 3 mmol) in glacial acetic acid (3 mL) was added 36% hydrogen bromide in acetic acid (3 mL). After standing at room temperature for 1 h, ether (50 ml) was added to the solution, and the precipitated hydrobromide was filtered off, dried in a desiccator over foeforpentoxide and sodium hydroxide for 2 hours, then dissolved in methanol (20 ml) and deienized on medium baex anion exchanger Wofatit L 150. in methanol.

The methanolic eluate was evaporated, and the residue was dried by azeotropic distillation from a mixture of methanol-benzene and tetrahydrofuran-benzene. The residue was dissolved in dimethylformamide (5 mL) and succinic anhydride (600 mg) was added and the reaction mixture was heated at 80 ° C for 2 h.

The reaction solution was then evaporated in vacuo, the product was precipitated by the addition of water (5 ml) and filtered. Crystallization from 2-pranol and water gave 720 mg (48%) of the product. The sample to be analyzed was crystallized similarly, m.p. 189-191 ° C.

Example 2 Benzyloxycarbonylglycyl-S-benzylcysteine p-nitroanilide

Prepared in a similar manner to the corresponding benzyloxycarbonyl derivative of Example 1. Crystallization from ethyl acetate and petroleum ether gave the product in 68% yield, m.p. 95 to 97 ®C.

3-Carboxypropionylglycyl-5-benzylcyeline p-nitroanilide

It was prepared in a similar manner to the corresponding 3-carboxypropionylderivative shown in Example 1. Crystallization from 2-propanol and water gave the product in 51% yield, m.p.

Mp 182-183 ° C.

Example 3 Benzyloxycarbonylglycyl-glycyl-S-benzyl-cysteine p-nitroanilide

It was prepared in a similar manner to the corresponding benzyloxycarbonyl derivative shown in Example 1, from the corresponding benzyloxycarbonylglycylglycine and S-benzylcysteine p-nitroanilide by the carbodiimide method. Crystallization from 2-propanol and petroleum ether gave the product in 65% yield and m.p. Mp 124-126 ° C.

The same product was obtained by the carbodiimide method from benzyloxycarbonylglycine and glycyl-S-benzylcyeteine p-nitroanilide obtained according to Example 2; m.p. Mp 125-127 ° C.

3-Carboxypropionylglycyl-glycyl-S-benxylcysteine p-nitreanilide

It was prepared in a similar manner to the corresponding 3-carbexypropionylderivative shown in Example 1. Crystallization from dimethylformamide and 2-propanol gave the product in 53% yield, mp 182-184 ^e C.

Example 4 Benzyloxycarbonylleucyl-leucyl-S-benzylcysteine p-nitroanilide

To a solution of benzyloxycarbonylleucyl-leucine hydrazide (3.95 g; 10 mmol) in tetrahydrofuran (80 mL) and 20% hydrochloric acid (4 mL) cooled to -15 ° C was added a solution of sodium nitrite (700 mg) in water (2 mL). , 8 ml). After 8 min (stirring and cooling (-10 ° C), pre-cooled (-15 ° C) ethyl acetate (150 mL) was added to the reaction solution) and after stirring for 2 min, the organic phase was shaken with a pre-cooled (saturated) sodium bicarbonate solution. 8% sodium chloride solution, dried over anhydrous sodium sulfate and added to a solution of S-benzylcysteine nitroanilide (3.30 g; 10 mmol) in dimethylformamide (30 mL).

After standing at +3 ° C for 12 h, the reaction solution was evaporated in vacuo and the residue was precipitated with water (50 mL); After standing at +3 ° C for 12 h, the product was filtered off, washed with water and crystallized from boiling ethanol (40 ml) and water (200 ml). 5.6 g (81%) of the product with m.p. M.p. 148-150 ° C.

3-Carboxypropionylleucyl-leucyl-S-benzylcysteine p-nitreanilide

It was prepared in a similar manner to the corresponding 3-carboxypropionylderivative shown in Example 1. Crystallization from 2-propanol and water gave the product in 54% yield, m.p.

Mp 160-162 ° C.

Example 5 p-Nitreanilide target. butyloxycarbonylalanyl-methionyl-S-benzylcysteine

It was prepared in a similar manner to the corresponding benzylexcarbonyl derivative from p-nitreanilide S-benzylcysteine and the corresponding target. butyloxycarbonylalanyl-methionine by the kerbodiimide method in 82% yield and m.p. Mp 129-132 ° C.

3-Carbexypropionylalanyl-methionyl-S-benzylcysteine p-nitroanilide

The target was removed by treatment with 211 hydrogen chloride in acetic acid. the butyloxycarbonyl group, the hydrochloride obtained, was deionised on Wefatit L 150 and worked up as described in Example 1. Crystallization from ethyl acetate yielded the product in 52% e. 139 to 142 ®C.

Example6 Benzyloxycarbonylglycyl-leucyl-S-benzylcysteine p-nitroanilide

It was obtained by the azide method from the corresponding benzyloxycarbonylglycyl-leucine hydrazide and S-benzylcysteine p-nitroanilide as described in Example 4 in a yield of 6.9% and m.p. Mp 177-180 ° C.

3-Carboxypropionylglycyl-leucyl-S-benzyloysteine p-nitroanilide

It was prepared in a similar manner to that described in Example 6 in a yield of 52% and m.p. 126 DEG-129 DEG.

Example 7 Benzyloxycarbonylleucyl-alanyl-S-benzylcysteine p-nitroanilide

Prepared by the azide method from the appropriate benzyloxycarbonylleucyl-alanine hydrazide and S-benzylcysteine p-nitroanilide as shown in Example 4, in a yield of 71% and m.p. Mp 168-172 ° C.

3-Carboxypropionylleucyl-alanyl-S-benzylcysteine p-nitroanilide

It was prepared in a similar manner as in Example 1, in a yield of 49,556 and mp 205-208 ^° C.

Example 8 Benzyloxycarbonylleucyl-S-benzylcysteine p-nitroanilide

It was prepared by carbodilmld method from benzyloxycarbonyllaucine and S-banzylcysteine p-nitroanilide as described in Example 1, in a yield of 5556 and m.p. M.p. 141-143 ° C.

3-Kerboxyproplonyllucyl-S-benzylcysteine p-nitroanilide

It was prepared in a similar manner as in Example 1, in a yield of 56.56 and m.p. Mp 189-192 ° C.

Claims (5)

Arylamides of dipeptides or tripaptides and a C-terminal S-aralkylcysteine radical of formula 1 X - A - B - Cys (Y) - KH - Z (I) wherein X represents an alkanecarbonyl radical of Cg to Cg, - a carbozyalkanecarbonyl radical of up to Cg, A is a residue of glycine, leucine, phenylalanine, methionine or zero, B is a residue of glycine, bacon, leucine, phenylalanine or methionine, Y is benzyl or p-nitrobansyl; and Z is p-nitrophenyl. 2. A process for the preparation of the compounds of the formula (I) according to claim 1, characterized in that the compound of the formula (II) is first used Cys (Y) - NH - Z (II), in which Y and Z denote the same as in formula I, condenses with a protected compound VA or WAB, wherein A and B denote the same as in a formula I and W protecting group, for example benzyloxycarbonyl, after which the remainder of the protecting group W is introduced into the prepared intermediate, where X is the same as in formula I. 3. A process for the preparation of a compound of formula (I) according to claim 1, characterized in that the compound of formula (II) Cys (Y) -NH-Z (II), wherein Y and Z are the same as in Formula I, condensed and protected with compound XA or XAB, wherein X and A and B are the same as in Formula I to form a compound of Formula I . 4. A process for the preparation of the compounds of formula (I) according to claim 1, characterized in that the compound of formula (II) is first used Cys (Y) - NH - Z (II), in which Y and Z mean the same as in formula I, condenses with a compound of formula III (III), 7244022 wherein B denotes the same as in formula I and W a protecting group, for example benzyloxycarbonyl, whereupon the resulting compound of formula IV is formed W - B - Cys (Y) - NH - Z (IV), in which B and Ϊ and Z denote the same as in formula I and W the same as in formula III, after deprotection W, condenses with a compound of formula V W - A (7), wherein A denotes the same as in formula I and W denotes the same as in formula III, after the removal of the protecting group W, a residue X with the meaning given in formula I is incorporated into the resulting peptide. 5. A process for the preparation of a compound of formula (I) according to claim 1, characterized in that the compound of formula (II) Cys (Y) - NH - Z (II), wherein Y and Z are the same as in Formula I, is condensed with a compound of formula 71 X-B (VI), or with a compound of formula 711 X - A - VII (VII), in which X and A and B are the same as in formula I, to form a compound of formula I.