JPH0822842B2 - Arginyl-3-tert-alkyloxycarbonyl-4-nitroanilide - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (1) Field of Industrial Application The present invention provides structural formula (I) [In the formula, R ₁ , R ₂ and R ₃ are the same or different and are — (CH ₂ ) _n CH ₃ (n = 0 to 3). ] Arginyl-3-tert-alkyloxycarbonyl-4-nitroanilide and its acid addition salt shown by these.

INDUSTRIAL APPLICABILITY The compound (I) of the present invention is useful as a raw material for synthesizing a chromogenic substrate for measuring enzyme activity, and in particular, has specificity for hydrolyzing the carboxyl group 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 the enzymatic activity of enzymes.

(2) Conventional Technology 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 chromogenic compound by the action of the enzyme. In this method, the enzyme and the substrate are reacted to dissociate the chromophoric compound, and the absorbance of light of a certain wavelength is measured to quantify the target enzyme.

The enzyme activity measurement substrate used in such an assay method is
It is required to have high sensitivity to enzyme, specificity, easy detection of decomposed products, and good solubility in water or buffer solution.

JP-A-59-106446 discloses a compound of formula (II) as a substrate for enzyme activity of thrombin, kallikrein, urokinase, plasmin, etc. A chromogenic substrate represented by the formula (wherein X is H or a protecting group generally used for peptide synthesis, A and B are amino acids or derivative residues thereof) has been proposed. R is -O
The ester type of C _n H _{2n + 1} , the amide type of -NHC _n H _{2n + 1} , or the one of amino acid residue is described, but the carboxyl type of R = OH is described. Absent.

(3) Problems to be Solved by the Invention It is considered that the carboxyl type substrate for measuring enzyme activity is preferable to the ester type and amide type substrates from the viewpoint of solubility in water or a buffer solution, and the carboxyl type is used. Therefore, it is possible that specificity for the enzyme may occur, so the development of the same type of substrate is desired.
It is extremely difficult to synthesize for the following reasons.

The above-mentioned substrates are synthesized by the reaction represented by the following reaction formula as shown in JP-A-59-106446.

In the above reaction, when the compound (b) in which R = OH is used to obtain a carboxyl type substrate, a by-product is generated due to the presence of the carboxyl group, and the target substrate is obtained in high yield. Have difficulty. In order to prevent side reactions, it is necessary to protect the carboxyl 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 an ester group must be removed later. However, in the case of esters or amides of primary or secondary alcohols such as n-butyl ester, isobutyl ester, methylamide, ethylamide and the like described in the above-mentioned published patent specifications, the operation for removing such a protecting group is Ester of primary or secondary alcohols, because other peptide bonds in the substrate molecule may be cleaved,
Or the protecting group which is an amide cannot be removed.

An object of the present invention is to solve the above-mentioned problems and provide an intermediate capable of synthesizing a substrate of R = OH in the above formula (II).

(4) Means for solving the problem Under these circumstances, as a result of our earnest research, the formula (I) [In the formula, R ₁ , R ₂ and R ₃ are the same or different and are — (CH ₂ ) _n CH ₃ (n = 0 to 3). ] It was found that the novel compound (arginyl-3-tert-alkyloxycarbonyl-4-nitroanilide) or acid addition salt thereof represented by the following formula is effective for achieving the above object, and has completed the present invention. .

In the above formula (I), For example, the following may be mentioned.

The novel compound of formula (I) may be an acid addition salt, and examples of such an acid addition salt include hydrochloride, hydrobromide,
There are inorganic acid salts such as phosphates, sulfates and nitrates; organic acid salts such as succinate, malate, citrate, lactate and benzenesulfonate.

The novel compound of formula (I) or an acid addition salt thereof is extremely useful as an intermediate for synthesizing a carboxyl-type enzyme activity measuring substrate. That is, as shown in Reference Example 1 described later, a dipeptide or a derivative thereof, such as D-γ-
(3-pentyloxy) -glutamyl-glycine and a compound of formula (I), for example The compound whose partial tert-alkyl ester is a t-butyl ester is reacted and then the tert-alkyl of the resulting compound is
By removing the alkyl ester by acidolysis, a novel carboxyl-type enzyme activity measurement substrate can be easily obtained in high yield.

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

The DCC method which is often used in the peptide synthesis of N ^α- t-butyloxycarbonyl-arginine (III) hydrate and 5-amino-2-nitrobenzoic acid-tert-alkyl ester (IV) in the first step Is dehydrated and condensed with to obtain N ^α- t-butyloxycarbonyl-arginyl-3-tert-alkyloxycarbonyl-4-nitroanilide (V).

Next, in the second step, N ^α- t-butyloxycarbonyl-arginyl-3-tert-alkyloxycarbonyl-4-nitroanilide (V) is hydrolyzed to protect t-butyl which protects the α-amino group of arginine. Only the oxycarbonyl (BOC) group is removed by acid decomposition. The problem here is that in the commonly used BOC group elimination method, t
The target compound cannot be obtained because the tert-alkyl ester such as -butyl ester is also cleaved. In order to solve this point, as a result of diligent studies, as a result of elimination of the protecting group in the presence of hydrochloric acid, acetic acid and dimethylformamide, preferably 2N hydrochloric acid-acetic acid-dimethylformamide, only the BOC group was removed. It was found that it can be selectively desorbed.

Thus, using a compound such as the compound of the formula (V) in which the α-amino group of the arginine moiety is protected by a BOC group and has a tert-alkyl ester such as t-butyl ester, this can be carried out under specific conditions. The compound of formula (I) of the present invention can be synthesized for the first time by subjecting it to the elimination reaction of the protecting group. Accordingly, such a compound of formula (V), and a compound of such formula (V), in the presence of hydrochloric acid, acetic acid and dimethylformamide, preferably 2N hydrochloric acid-acetic acid-
It is also an object of the present invention to provide a method for obtaining a compound of formula (I) by deprotection in the system of dimethylformamide.

In the above method, in addition to the case where the protective group for the arginine moiety is a BOC group as the compound of formula (V), 1,1-dimethylpropyloxycarbonyl group, 1,1-dimethylbutyloxycarbonyl group, 1,1 It is also applicable to the compound of the formula (V) in the case of an ester of an alcohol having a tertiary carbon such as a dimethylpentyloxycarbonyl group and a 3-ethylpentyl-3-yloxycarbonyl group.

(5) Effects of the Invention As described in detail above, the feature of the present invention is that the compound of the formula (I) is excellent as a starting material for the synthesis of the carboxyl type enzyme activity measuring substrate represented by the formula (II). Where it is.

For example, a carboxyl type compound represented by the formula (II) is prepared by using a raw material with no carboxyl group protection, arginyl-3-
It can be generally said that peptide synthesis tends to cause a side reaction when carboxy-4-nitroanilide is used as a starting material for the synthesis. Therefore, it is difficult to obtain the desired carboxyl type compound in high yield. Further, when a compound in which a carboxyl group is protected as an ester such as methyl, ethyl or isobutyl is used as a raw material, the ester is hydrolyzed in order to finally eliminate the ester to give a carboxyl type target compound. Since it requires manipulation, it adversely affects the peptide chain. Alternatively, when a raw material protected by a protective group that can be eliminated by reduction such as benzyl ester is used, the nitro group in the substrate is also reduced during the reduction, and thus such raw material cannot be used. In the compound (I) of the present invention using a tert-alkyl ester such as t-butyl ester as a protecting group for the raw materials using these protecting groups, the side group is suppressed because the carboxyl group is protected, Moreover, since such a protecting group can be easily removed, the peptide of Reference Example 1 is not adversely affected without adversely affecting the peptide chain and the finally obtained substrate.
As shown in (1), it is possible to obtain a target product, which is a carboxyl-type enzyme activity measurement substrate, in high yield.

For example, the carboxyl-type substrate for enzyme activity measurement D-γ- (3-pentyloxy) -glutamyl-glycyl-arginyl-3-carboxy-4-, which is obtained from the compound (I) of the present invention as a raw material and is obtained in Reference Example 1, As shown in Reference Example 2, nitroanilide (VI) dihydrochloride was used as CHR-TRY.
When used as a control (Pentapharm), the reactivity to trypsin is 1.88 times and the reactivity to normal serum is 1/20.
And is excellent in its 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 the carboxyl type enzyme activity measurement substrate represented by the formula (II).

Furthermore, a substrate for measuring a carboxyl-type enzyme activity obtained from the compound (I) of the present invention has reactivity with an enzyme,
Extremely excellent in specificity.

(6) Examples Hereinafter, the synthesis method of the compound (I) of the present invention will be specifically described with reference to Examples.

Example 1 (i) N ^α -t-butyloxycarbonyl-arginyl-3-t-butyloxycarbonyl-4-nitroanilide (V) (in the formula (V), R ₁ , R ₂ and R ₃ are methyl). Compound) Synthesis of N ^α- t-butyloxycarbonyl-arginine (II
I) 27.41 g (83.4 mmol) of hydrochloride / hydrate and 5-amino-2-nitro-benzoic acid-t-butyl ester (IV)
(Compound in which R ₁ , R ₂ and R ₃ in the formula (IV) are methyl) 1
After dissolving 9.87 g (83.4 mmol) in 167 ml of anhydrous pyridine, a solution of 37.86 g (183.5 mmol) of N—N′-dicyclohexylcarbodiimide in 83 ml of pyridine was added dropwise under cooling with stirring at −5 ° C., and the mixture was added. Reaction was carried out at room temperature with stirring overnight. After completion of the reaction, 333 ml of ethyl acetate was added to the reaction solution, and the precipitated dicyclohexylurea was filtered off,
The solvent was distilled off under reduced pressure, 750 ml of ethyl acetate was added thereto, and the insoluble matter was collected by filtration to obtain 37.32 g (yield 84.3%) of N ^α
-T-butyloxycarbonyl-arginyl-3-t-
Butyloxycarbonyl-4-nitroanilide (V) was obtained.

(Ii) Arginyl-3-t-butyloxycarbonyl-
4-nitroanilide (I) (in the formula (I), R ₁ , R ₂ , R ₃
(Compound in which is methyl) 7.97 g (15 mmol) of N ^α -t-butyloxycarbonyl-arginyl-3-t-butyloxycarbonyl-4-nitroanilide (V) obtained here was added to 9 ml of DMF and 3 ml of acetic acid.
Dissolve in water, add 60 ml of 2N hydrochloric acid-acetic acid under stirring with ice-cooling, and bath temperature
The reaction was carried out at 15 ° C for 30 minutes to selectively remove the N ^α protecting group. After the reaction was completed, 36 ml of ethyl acetate was added to the reaction solution, and the solution was poured into 2.5 l of ether to obtain a precipitate,
The crystals were collected by filtration and dried under reduced pressure to obtain 6.48 g of crude crystals. The crystals were further purified by a Dowex 2 × 8 (acetic acid type) [Dow Chemical] column (eluent: methanol), and an equivalent amount of 0.1N hydrochloric acid-methanol was added, followed by precipitation with ether to give arginyl-3-t-. 5.68 g of crystals of butyloxycarbonyl-4-nitroanilide (I) dihydrochloride (yield 81.0
%) Was obtained.

Melting point 65-95 ° C (decomposition) Specific optical rotation ▲ [α] ²⁵ _D ▼ = + 41.5 ° C (C = 1, water) This crystal is 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: Measured value (%) as C ₁₇ H ₂₈ N ₆ O ₅ Cl ₂ · 1 / 2H ₂ O C: 42.77 H: 6.20 N: 17.60 Theoretical value (%) C: 42.86 H: 6.14 N: 17.64 ( 7) Reference Example As Reference Example 1 below, D-γ- (3-pentyloxy) -glutamyl-containing the compound (I) of the present invention as a raw material.
In addition to specifically explaining the synthesis of glycyl-arginyl-3-carboxy-4-nitroanilide (VI), as Reference Example 2, CHR-TRY (Pentapharm) was used as a control and a formula ( The measurement results of the reactivity of the compound of VI) with various enzymes and normal serum are shown.

Reference Example 1 Compound (I) dihydrochloride (2.34 g, 5 mmol) was added to DMF10.
After dissolving in ice water, under ice cooling, 0.65 ml of N-ethylmorpholine (5
Mmol), and the mixture was reacted with stirring for 5 minutes, and then t-butyloxycarbonyl-D-γ- (3-pentyloxy)
-Glutamyl-glycine-4,6-dimethylpyrimidyl-
A solution prepared by dissolving 248 g (5 mmol) of 2-thioester in 10 ml of DMF was added under ice-cooling, and the mixture was reacted with stirring at room temperature overnight. After the reaction was completed, DMF was distilled off under reduced pressure, and 100 ml of ethyl acetate was added to the residue.
Add 50 ml of cold 5% hydrochloric acid twice and 50 ml of saturated saline to 1
Wash twice with 50 ml of 10% aqueous sodium hydrogen carbonate solution and twice with 50 ml of saturated saline solution, dehydrate and dry over anhydrous magnesium, and distill off the solvent under reduced pressure. Sephadex LH-20 chromatography (eluent: methanol) 2.56 g of t-butyloxycarbonyl-D-γ- (3-pentyloxy) -glutamyl-glycyl-arginyl-3-t-butyloxycarbonyl-4-nitroanilide (VII) (yield 8
0.0%) was obtained.

2.42 g (3 mmol) of the compound (VII) obtained here was added to acetic acid.
The mixture was dissolved in 3 ml, 2N hydrochloric acid-acetic acid (15 ml, 30 mmol) was added dropwise under ice cooling, and the mixture was reacted at room temperature for 1 hour. 50 after reaction
The reaction solution was poured into 0 ml of ether, the precipitate was collected by filtration, and Toyopearl HW40F chromatography (eluent: 30% acetic acid) [Toyo Soda]
Purified by D-γ- (3-pentyloxy) -glutamyl-glycyl-arginyl-3-t-carboxy-
1.5 g of 4-nitroanilide dihydrochloride (VI) (yield 75
%)Obtained. The physical properties and analysis results are shown below.

Melting point 108-147 ° C (decomposition) Specific optical rotation ▲ [α] ²⁵ _D ▼ = -58.0 ° C (C = 1, water) This crystal is a silica gel thin layer chromatograph (n
− Butanol: acetic acid: water = 4: 1: 2) with a single spot (Rf
= 0.42) was given.

Elemental analysis value: C ₂₅ H ₄₀ N ₈ O ₉ Cl ₂ · 7/5 H ₂ O measured value (%) C: 43.44 H: 6.17 N: 16.13 Theoretical value (%) C: 43.34 H: 6.23 N: 16.17 Reference Example 2 1) Substrate solution: Each substrate solution was dissolved in water and used at 10 mM.

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

3) Enzyme used 4) Reaction stop solution: 10% acetic acid aqueous solution 5) Measurement method a) Various enzyme buffer 0.5 ml and substrate solution 0.1 ml were collected in a silicon-treated hard glass test tube or plastic test tube, and 37 ° C.
Preheat for 10 minutes in a constant temperature bath.

Then, 0.05 ml of enzyme reagent was added and the enzyme reaction was performed at 37 ° C for 10
Conduct for minutes.

Exactly 10 minutes later, 2.5 ml of the reaction stop solution is added to stop the enzymatic reaction, and the mixture is allowed to stand at 37 ° C for 10 minutes, and then the absorbance at 405 nm is measured.

b) Normal serum 0.5 ml of the buffer solution and 0.1 ml of the substrate solution were collected in a silicon-treated hard glass test tube or plastic test tube at 37 ° C.
Preheat for 5 minutes in a constant temperature bath.

Then, 0.1 ml of normal serum was added to the enzyme reaction at 37 ° C for 5
Conduct for minutes.

Exactly 5 minutes later, 2.0 ml of the reaction stop solution was added to stop the enzyme reaction, and the mixture was allowed to stand at 37 ° C for 10 minutes, and then the absorbance at 405 nm was measured.

6) Measurement result (absorbance of a is 1) Abbreviation Z: benzyloxycarbonyl Glu: Glutamic acid Gly: Glycine Arg: Arginine PNA: P-nitroaniline 5ANBA: 5-amino-2-nitrobenzoic acid HCl: hydrochloric acid As is clear from the above measurement results, the compound of the present invention ( The reactivity of compound (VI) obtained from I) to trypsin is 1.88 times that of CHR-TRY, and the reactivity to normal serum is 2
It is 1/0, and the compound (VI) is excellent in reactivity and specificity as a substrate for measuring trypsin activity.

Claims (1)

[Claims] 1. The following structural formula (I) [In the formula, R ₁ , R ₂ and R ₃ are the same or different and are — (CH ₂ ) _n CH ₃ (n = 0 to 3). ] Arginyl-3-tert-alkyloxycarbonyl-4-nitroanilide and its acid addition salt shown by these.