JPH023660A - Arginyl-3-tert.-alkyloxycarbonyl-4-nitroanilide - Google Patents

Abstract

NEW MATERIAL:A compound expressed by formula I [R1, R2 and R3 are -(CH2)nCH3; n is 0-3] and acid addition salt thereof. EXAMPLE:Arginyl-3-tert.-butyloxycarbonyl-4-nitroanilide. USE:A synthetic raw material for coloring substrate for measuring enzyme activity, especially useful as a synthetic raw material for a coloring substrate for measuring enzyme activity of an enzyme having a specificity capable of hydrolyzing carboxyl side of a basic amino acid such as trypsin, urokinase or thrombin. PREPARATION:N<a>-tert.-Butyloxycarbonyl-arginine.hydrate expressed by formula II is subjected to condensation through dehydration with a 5-amino-2-nitrobenzoic acid-tert.-alkyl ester expressed by formula III according to DDC method used in peptide synthesis and then the resultant condensed product is subjected to deprotecting using acetic acid in dimethylformamide to provide the compound expressed by formula I.

Description

DETAILED DESCRIPTION OF THE INVENTION (1) Industrial Application Field The present invention relates to structural formula (I) [wherein R1, R2, R3 are the same or different,
H2) nCH3 (n = 0-3). ] and its acid addition salt.

Compound (I) of the present invention is useful as a raw material for the synthesis of chromogenic substrates for measuring enzyme activity, and is particularly useful for enzymes that have the specificity of hydrolyzing the carboxy side of basic amino acids, such as trypsin, urokinase, and thrombin. It is useful as a starting material for the synthesis of chromogenic substrates for measuring enzyme activity.

(2) Prior Art As a method for quantifying enzymes such as trypsin, urokinase, and thrombin in human plasma, there is a method using a substrate that dissociates a color-forming compound by the action of the enzyme. In this method, an enzyme and a substrate are reacted to dissociate a color-forming compound, and the target enzyme is quantified by measuring the absorbance of light at a certain wavelength.

The substrate for enzyme activity measurement used in such a measurement method is required to have high sensitivity and specificity for the enzyme, easy detection of decomposition products, and good solubility in water or buffer.

JP-A-59-106446 describes the development of a substrate of the same type as the specificity of formula (II) as a substrate for the enzyme activity of thrombin, kallikrein, urokinase, plasmin, etc., which is expected to occur. It is extremely difficult to synthesize for these reasons.

The synthesis of the above-mentioned substrate is carried out by a reaction represented by the following reaction formula, as shown in JP-A-59-106446.

A chromogenic substrate represented by the formula (wherein X is H or a protecting group generally used in peptide synthesis, and A and B are amino acid or derivative residues thereof) has been proposed. R is an ester type of 10CnH2n+1, -NHCnH2n
+1 amide type or amino acid residue is described, but R=OH carboxyl type is not described.

(3) Problems to be Solved by the Invention Carboxyl-type substrates for measuring enzyme activity are considered to be more preferable than ester- or amide-type substrates in terms of solubility in water or buffer solutions, and carboxyl-type substrates are considered preferable to ester- and amide-type substrates. In the above reaction for the enzyme, the compound (b) in which R=OH is obtained in order to obtain a substrate in carboxyl form.
When using , by-products are generated due to the presence of the carboxy group, making it difficult to obtain the desired substrate in high yield. In order to prevent side reactions, it is necessary to protect the carboxy group as an ester, amide, etc. as shown in the above-mentioned published patent specifications. In this case, if a carboxyl type substrate is to be obtained, the protecting group such as the ester group must be removed afterwards. However, the first Is such as n-butyl ester, isobutyl ester, methylamide, ethylamide, etc. described in the above-mentioned published patent specification
In the case of esters or amides of L or 21-alcohols, there is a risk that other peptide bonds in the substrate molecule may be cleaved by the removal of such protecting groups. The protecting group, which is an amide, cannot be removed.

The present invention solves the above-mentioned problems and solves the above-mentioned equation (
The object of the present invention is to provide an intermediate capable of synthesizing a substrate in which R is a carboxyl type in II).

(4) Means for solving the problem As a result of our intensive research under these circumstances, we found that formula (I) R8 [wherein R1, Ll, and R3 are the same or different, -(
CH2)nCH3 (n = O~3). We have discovered that the novel compound represented by 1 (arginyl-3-tert-alkyloxycarbonyl-4-nitroanilide) or its acid addition salt is effective in achieving the above object, and have completed the present invention. Ta.

R1 R3 These include:

The novel compounds of formula (I) may be acid addition salts, such as inorganic acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, nitrates; succinic acid; salt,
Organic acid salts include malate, citrate, lactate, and benzenesulfonate.

The novel compound of formula (I) or its acid addition salt is extremely useful as an intermediate for synthesizing a carboxyl-type substrate for measuring enzyme activity. That is, as shown in Reference Example 1 described below, dipeptides or derivatives thereof, such as D-γ-(
3-pentyloxy)-glutamyl-glycine and the formula (I
), such as the first stage R.

J By reacting a compound that is a butyl ester and then removing the tert-alkyl ester of the resulting compound by hydrolysis, a novel carboxyl-type substrate for enzyme activity measurement can be easily obtained in high yield. .

The compound of formula (I) or its acid addition salt according to the present invention is
It can be synthesized according to the reaction formula shown below.

(V) Second step In the first step, N'-t-butyloxycarbonyl-arginine (III) hydrate and 5-amino-2-nitrobenzoic acid-tert-alkyl ester (IV) may be synthesized by peptide synthesis. Dehydrated and synthesized using the BOC method used,
NO-t-butyloxycarbonyl-arginyl-3-
tert-alkyloxycarbonyl-4-nitroanilide (V) is obtained.

Then, in the second step, NQ -t-butyloxycarbonyl-arginyl-3-tert-alkyloxycarbonyl-4-nitroanilide (V) is hydrolyzed to form 1-butyloxy which protects the α-amino group of arginine. Only the carbonyl (BOC) group is hydrolyzed and removed.

The problem here is that the commonly used method for eliminating the BOC group also cleaves tert-alkyl esters such as t-butyl ester, making it impossible to obtain the desired compound. In order to solve this problem, as a result of intensive studies, we removed only the BOC group by removing the protecting group in the presence of hydrochloric acid, acetic acid, and dimethyl sulfoxide, preferably under the conditions of 2N hydrochloric acid-acetic acid-dimethylformamide. It has been found that selective desorption is possible.

Therefore, using a compound such as the compound of formula (V) in which the α-amino group of the arginine moiety is protected with a BOC group and having a tert-alkyl ester such as t-butyl ester, it is possible to By subjecting it to a protecting group elimination reaction, it became possible to synthesize the compound of formula CI) of the present invention for the first time. Therefore, the compound of formula (V) and the compound of formula (V) are deprotected in the presence of hydrochloric acid, acetic acid and dimethylformamide, preferably in a 2N hydrochloric acid-acetic acid-dimethylformamide system to obtain a compound of formula (I). It is also an object of the present invention to provide a method for obtaining.

In the above method, 1,1-
It is an ester of alcohol with a tertiary carbon such as dimethylpropyloxycarbonyl group, 1,1-dimethylbutyloxycarbonyl group, 1.1-dimethylpentyloxycarbonyl group, 3-ethylbentyloxycarbonyl group, etc. It can also be applied to compounds of formula (V) etc.

(5) Effects of the Invention As detailed above, the feature of the present invention is that the compound of formula (I) is excellent as a starting material for the synthesis of the carboxyl type substrate for measuring enzyme activity represented by formula (II). It's where you are.

For example, a carboxyl type compound represented by formula (II) is used as a starting material with an unprotected carboxyl group, arginyl-3
When synthesis is performed using -carboxy-4-nitroanilide as a raw material, side reactions are likely to occur due to the presence of carboxyl groups, which is true for peptide synthesis in general. Therefore, it is difficult to obtain the desired carboxyl type compound in high yield. In addition, when a compound with a carboxyl group protected as an ester such as methyl, ethyl, or isobutyl is used as a raw material, hydrolysis of the ester is necessary to ultimately eliminate the ester and obtain a carboxyl-type target compound. Because it requires manipulation, it has a negative effect on the peptide chain. Alternatively, when a raw material protected with a protective group that can be removed by reduction, such as a benzyl ester, is used, such a raw material cannot be used because the nitro group in the substrate is also reduced during the reduction.

For raw materials using these protecting groups, t-
The compound (I) of the present invention using a tert-alkyl ester such as a butyl ester has a protected carboxyl group, so side reactions are suppressed, and such a protecting group can be easily removed. Reference Example 1 without adversely affecting the chain and the final substrate obtained.
As shown in the figure, the target product, which is a carboxyl-type substrate for measuring enzyme activity, can be obtained in high yield.

Carboxyl type enzyme activity measurement substrate D obtained using Compound (I) of the present invention as a raw material, for example, obtained in Reference Example 1
-γ-(3-pentyloxy)-glutamyl-glycyl-arginyl-3-carboxy-4-nitroanilide (
VI) dihydrochloride is CHR- as shown in Reference Example 2.
When TRY (Pentapharm) is used as a control, it has 1.88 times the reactivity to trypsin and 1/20th the reactivity to normal serum, and is excellent in reactivity and specificity as a substrate for trypsin.

As described above, it is clear that the compound (I) of the present invention is excellent as a starting material for a substrate for measuring the activity of a carboxyl-type enzyme represented by formula (II).

Furthermore, the carboxyl-type substrate for measuring enzyme activity obtained from the compound (I) of the present invention is extremely excellent in reactivity and specificity for enzymes.

(6) Examples The method for synthesizing compound (I) of the present invention will be specifically explained in Examples below.

Example 1 37.86 g of N'-dicyclohexylcarbodiimide
A solution of (183.5 mmol) in 83 rne of pyridine was added dropwise, and the mixture was stirred and reacted overnight at room temperature.

After the reaction was completed, ethyl acetate 333me was added to the reaction solution,
After filtering out the precipitated dicyclohexylurea, the solvent was distilled off under reduced pressure, 750 m4' of ethyl acetate was added thereto, and the insoluble matter was filtered out to give 37.32 g (yield: 84
．． 3%) of Nfl-t-butyloxycarbonyl-arginyl-3-t-butyloxycarbonyl-4-nitro-anilide (V) was obtained.

NG-t-butyloxycarbonyl-arginine (I
II) 27.41 g (83.4 mmol) of hydrochloride/hydrate and 5-amino-2-nitro-benzoic acid 7-butyl ester (■) (in formula (■), R, R2, R
Compound where 3 is methyl) 19.87 g (83,
After dissolving 4 mmol) in 167 m4' of anhydrous pyridine,
Cool to -5°C and stir, NQ -t-butyloxycarbonyl-arginyl-3-t-butyloxycarbonyl-4-nitro-anilide (V) obtained here.7.
97 g (15 mmol) in DMF 9 me and 3 ml acetic acid! Dissolved in 2N hydrochloric acid-acetic acid 60% while cooling with water and stirring.
Me was added and reacted for 30 minutes at a bath temperature of 15°C to selectively remove the N'' protective group. After the reaction was completed, 36mt' of ethyl acetate was added to the reaction solution, and the solution was diluted with 2°5e of ether. 6.48 g of crude crystals were obtained by pouring the precipitate into the solution, which was collected by filtration and dried under reduced pressure.
x2 x 8 (acetic acid type) [Dow Chemical]
Purified by column (eluent methanol) and an equivalent amount of 0.
5. Crystals of arginyl-3-t-butyloxycarbonyl-4-nitroanilide (I) dihydrochloride were obtained by adding IN hydrochloric acid-methanol and precipitating with ether.
68 g (yield 81.0%) was obtained.

Melting point: 65-95°C (decomposed) Specific optical rotation: [α]D = +41.5° (C = 1, water) This crystal was analyzed by silica gel thin layer chromatography (n-butanol:acetic acid:water = 4:1:2 ) with a single spot (Rf
:0.48) was given.

Elemental analysis value: C07H28N60. Ce2・112H2
Actual value (%) as 0 C: 42.77H: 6.2O
N: 17.60 theoretical value (%) C: 42.86H26
．． 14 N: 17.64 (7) Reference Example Below, as Reference Example 1, D-γ-(3-pentyloxy)-glutamyl-glycyl-arginyl-3-carboxy- made from the compound (I) of the present invention The synthesis of 4-nitroanilide (VI) will be specifically explained, and as Reference Example 2, CHR-TRY (Pentapharm)
The results of measuring the reactivity of the compound of formula (Vl) as a substrate for enzyme activity measurement with various enzymes and normal serum are shown below.

Reference Example 1 2.34 g (5 mmol) of compound (I) dihydrochloride was added to D
After dissolving in MFlome, 0.65 me (5 mmol) of N-ethylmorpholine was added dropwise under water cooling, and after reacting with stirring for 5 minutes, t-butyloxycarbonyl-D-'γ-
(3-pentyloxy)-glutamyl-glycine-4゜6-dimethylpyrimidyl-2-thioester 248g (
A solution of 5 mmol) dissolved in 10 ml of DMF was added under water cooling, and the mixture was stirred and reacted overnight at room temperature. After the reaction was completed, DMF was distilled off under reduced pressure, 100me of ethyl acetate was added to the residue, and the mixture was diluted with 50me of cold 5% hydrochloric acid twice, once with 50me of saturated brine, twice with 50me of 10% sodium bicarbonate aqueous solution, and saturated brine. Washed twice with 50ml of water, dehydrated and dried over anhydrous magnesium, distilled off the solvent under reduced pressure, purified with Sephadex LH-20 chromatography (eluent: methanol), and purified with t-butyloxycarbonyl-D-γ- (3-pentyloxy)-glutamyl-glycyl-arginyl-3-
t-Butyloxycarbonyl-4-nitroanilide (■
) 2.5 eg (yield $80.0%) was obtained.

2.42 g (3 mmol) of the compound (■) obtained here was dissolved in 3 me of acetic acid, and mixed with 2N hydrochloric acid-acetic acid (15 m?) under water cooling.
(30 mmol) was added dropwise and reacted at room temperature for 1 hour.

After the reaction was completed, the reaction solution was poured into 500me ether, the precipitate was collected by filtration, and the precipitate was collected using a Toyover ()L/HW40F chromatograph (
Eluent: 30% acetic acid) [Toyo Soda] to purify D-
γ-(3-pentyloxy)-glutamyl-glycyl-
1.5 g (yield 75%) of arginyl-3-t-carboxy-4-nitroanilide dihydrochloride (VI) was obtained. The physical properties and analysis results are shown below.

Melting point: 108-147°C (decomposed) Specific rotation: [α]D knee 58.0° (C=1, water) This crystal was analyzed by silica gel thin layer chromatography (n-butanol:acetic acid:water=4:1: 2) with a single spot (R
f=0.42).

Elemental analysis value: C25H4oN8o9Ce2・715H2
Actual value (%) as 0 C: 43.44H: 6.17
N: 16.13 theoretical value (%) C: 43.34 H: 6
．． 23N:16.17 Reference Example 2 1) Substrate solution: Each substrate solution was dissolved in water and used at 10 mM.

2) Buffer: buffer species, NaC (and their concentrations,
The pH (25°C) was determined as follows depending on the enzyme.

3) Enzyme used 4) Reaction stop solution = 10% acetic acid aqueous solution 5) Measurement method a) Add 0.5 ml of various enzyme buffers and 0.1 me of substrate solution to a silicon-treated hard glass test tube or plastic test tube. Collect and prewarm for 10 minutes in a 37°C constant temperature bath.

Then, 0.05 me of enzyme reagent was added to incubate the enzyme reaction for 3
Perform for 10 minutes at 7°C.

After exactly 10 minutes, add 2.5 mL of reaction stop solution to stop the enzyme reaction, and leave it at 37°C for 10 minutes.
Measure the absorbance in nm.

b) Collect 0.5 me of normal serum buffer and 0.1 rne of substrate solution into a siliconized hard glass test tube or plastic test tube, and prewarm in a 37°C thermostatic bath for 5 minutes. Next, 0.1 me of normal serum was added and the enzyme reaction was carried out at 370 ml.
0 for 5 minutes.

After exactly 5 minutes, stop solution 2. After adding Ome to stop the enzyme reaction, leave it at 37°C for 10 minutes, then 405 nm.
Measure the absorbance of

6) Measurement results (assuming the absorbance of a to 1) a: CH
R-TRY (Pentapharm) Z-Val
-Gly-Arg-PNA-HC (b: Compound ■ 2HC4' -H-D -Glu (-00H(C2H
3)2)-Gly-Arg-5ANBA Abbreviation 2: Penzyloxycarbonyl Glu: Glutamate G1y Nigericin Arg: Arginine PNA: P-Nitroaniline 5ANBA: 5-Amino-2-nitrobenzoic acid HC (:
Hydrochloric acid As is clear from the above measurement results, the compound of the present invention (
The reactivity of compound (VI) obtained from I) with trypsin is 1.88 times that of CHR-TRY, and the reactivity with normal serum is 1/20. It has excellent properties and specificity.

Claims (1)

[Claims] The following structural formula (I) ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I) [In the formula, R_1, R_2, and R_3 are the same or different, -(CH_2)_nCH_3 (n=0 to 3 ). ] Arginyl-3-tert-alkyloxycarbonyl-4-nitroanilide and its acid addition salt.