JPH07242639A - Determination of esterase or protease and reagent composition to be used therefor - Google Patents

Abstract

PURPOSE:To detect an esterase or protease, especially esterase existing in leukocyte by the development of a color (purple to blue) insusceptible to the coexisting substances by using a specific compound as a substrate. CONSTITUTION:Esterase or protease is determined by using a compound of formula I [A is amino-protected amino acid residue or peptide residue having protected N-terminal amino group; R is a (substituted)aryl; X is O, S or a (substituted)imino], preferably a compound of formula II (R' is an aryl, an alkoxy-substituted aryl or a halogen-substituted aryl; X' is S or imino) as a substrate.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for measuring ester degrading enzymes or proteolytic enzymes such as leukocyte esterase and a reagent composition used therefor.

[0002]

BACKGROUND OF THE INVENTION The detection of white blood cells in urine serves as an index for diagnosing urinary tract infections and renal diseases. Conventionally, urinary sediment microscopy has been widely used, but this method requires labor and time and requires a skilled person. There was a problem that On the other hand, in recent years, a simple test piece for detecting white blood cells utilizing the esterase activity in white blood cells has been found, and is partially put to practical use.
In this method, the ester compound used as a substrate is hydrolyzed by leukocyte esterase to generate an alcohol (phenol) component, and this is directly colored, or a diazonium salt is allowed to coexist with an alcohol (phenol There is a method of utilizing a reaction in which a (phenol) component is coupled with a diazonium salt to develop a color,
As the ester compound used as a substrate, in the case of the former method, sulfonephthalein esters (Japanese Patent Laid-Open No. 55-2
992), azo dye esters (Japanese Patent Publication No. 61-42749), and the latter method includes phenoxy-amino acid esters (Japanese Patent Publication No. 61-45982) and indoxyl esters (Japanese Patent Publication No. 61-45982). Japanese Patent Publication No. 2-43480), phenylpyroxyl esters (Japanese Patent Publication No. 62-41710) and the like are known.

However, all of the methods using these substrates have the drawbacks that the reaction takes a long time and the detection sensitivity is low. In order to overcome this drawback, attempts have been made to improve the reaction rate by adding an esterase reaction accelerator, but sufficient sensitivity has not yet been obtained. In addition, in the method using indoxyl ester or phenylpyroxyl ester, which is a substrate that is currently in practical use, since the color tone is reddish purple and shallow, it is easily affected by coexisting substances with colorability such as hemoglobin and bilirubin. In addition, in the production of these substrates, there is a problem in that the intermediate is unstable during the production, the operation is complicated, the yield is low, and the production cost is high.

[0004]

SUMMARY OF THE INVENTION The present invention has been made in view of the above situation, and can detect leukocytes rapidly and with high sensitivity in a deep color tone of purple to blue which is hardly affected by coexisting substances. Easy and cheap to manufacture,
It is intended to provide a reagent composition for measuring esterolytic enzyme or proteolytic enzyme.

[0005]

The present invention has the general formula [I]

[Chemical 4] [In the formula, A is an amino acid residue whose amino group is protected or N
The terminal amino group represents a protected peptide residue, R represents an aryl group which may have a substituent, and X represents an oxygen atom, a sulfur atom or an imino group which may have a substituent. . ] It is invention of the measuring method of esterolytic enzyme or proteolytic enzyme characterized by using the compound shown by these as a substrate. The present invention also provides a compound represented by the general formula [I]

[Chemical 5] [In formula, A, R, and X are the same as the above. ] It is invention of the reagent composition for measuring esterolytic enzyme or proteolytic enzyme characterized by including the compound shown by these, a diazonium salt, and a buffer substance. Furthermore, the present invention is an invention of a test piece for detecting leukocytes, which comprises the reagent composition held on an absorptive carrier or a film. Furthermore, the present invention has the general formula [II]

[Chemical 6] [In the formula, R'represents an aryl group, an alkoxy-substituted aryl group or a halogen-substituted aryl group, and X'represents a sulfur atom or an imino group. ] It is an invention of the compound shown by these.

That is, the inventors of the present invention have conducted extensive studies to achieve the above object, and as a result, by using the compound represented by the general formula [I], it is present in esterases or proteinases, especially in leukocytes. The present inventors have found that esterase can be detected with high sensitivity in a deep color tone (purple to blue) that is not easily affected by coexisting substances, and arrived at the present invention. Since these compounds are stable intermediates, many of them are easy to produce and have a high yield, and the production cost is necessarily low.

In the compound represented by the general formula [I] according to the present invention, the amino group represented by A is protected as an amino-protecting group of an amino acid residue or an N-terminal amino group-protected peptide residue. May have a substituent such as an N-benzyloxycarbonyl group or an N- (t-butoxycarbonyl) group, and may have a substituent such as an alkyloxycarbonyl group, a benzenesulfonyl group or a tosyl group. Amino acids that are well known in the art, such as a good arylsulfonyl group, a sulfenyl group which may have a substituent, a phosphoryl group which may have a substituent, and a carbamoyl group which may have a substituent. Protecting groups may be mentioned. As the amino acid residue, any of its L-type, D-type and racemic type can be used, but the L-type is particularly preferable, and glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine and the like can be used. Amino acid residues are particularly preferred. As the peptide residue, those in which 2 to 5 of the above amino acids are bound are preferable.

In the general formula [I], the substituent of the optionally substituted aryl group represented by R is a halogen atom such as a fluorine atom, a chlorine atom, a bromine atom or an iodine atom, for example, Alkoxy group such as methoxy group, ethoxy group, propoxy group, butoxy group (may be linear or branched), for example, alkylamino such as methylamino group, ethylamino group, propylamino group, butylamino group Group (may be linear or branched), for example, dialkylamino group such as dimethylamino group, diethylamino group, dipropylamino group, dibutylamino group (may be linear or branched) ), For example, formamino group, acetamido group, propionamido group, butyrylamino group, and other acylamino groups (which may be linear or branched), cyano groups, nitro groups, etc. Rukoto can, as the aryl group include a phenyl group, a tolyl group, a naphthyl group, methylnaphthyl group, and the like. The substituent of the imino group which may have a substituent represented by X is, for example, an alkyl group such as a methyl group, an ethyl group, a propyl group or a butyl group (in a straight chain or a branched chain, ), For example, aryl groups such as phenyl group, tolyl group and naphthyl group, and heterocyclic groups such as pyrrole group and pyridyl group.

In the compound represented by the general formula [II] according to the present invention, the aryl group represented by R ', the alkoxy-substituted aryl group and the halogen-substituted aryl group include, for example, phenyl group, tolyl group, Examples of the alkoxy group include naphthyl group and methylnaphthyl group.
For example, a methoxy group, an ethoxy group, a propoxy group, a butoxy group and the like (which may be linear or molecular),
Examples of the halogen atom include fluorine, chlorine, bromine and iodine.

The compound represented by the general formula [I] can be produced generally as follows. That is, first, the general formula [III]

[Chemical 7] [In the Formula, R and X are the same as the above. ] The 3-hydroxy thiazole derivative, the 3-hydroxy oxazole derivative or the 3- hydroxy imidazole derivative represented by the formula] is, for example, Acta Chemica Scandinavica, Vol. 17, p. 144, 1963.
Year, Journal of American Chemical Society, Vol. 97, 72.
97 pages, 1975, Acta Chemica Scandinavica, Volume 7, 1030.
Page, 1953, and the book by JVMetzger, "The Chemistry.
of Heterocyclic Compounds "Volume 34, Thiazole and Its
Derivative, Part 2, 1979, Published by John Wiley & Sons, IJ
Turchi, ibid., Volume 45, Oxazoles, 1986, and K. Hofmann.
It is synthesized according to a method known per se described in Ibidazole and Derivatives, 1953, Ibid. Then, the compound represented by the general formula [III] and the amino group-protected peptide or amino group-protected amino acid or a suitable reactive derivative thereof (eg, acid chloride, chloroformic acid ethyl ester, active ester). Etc.) for example, “Peptide Synthesis” by Izumiya et al., Maruzen Publishing, 1975, Houben-Weyl “Met.
hoden der Organischen Chemie "Volume 25/1, 2 1974
If the reaction is carried out in a conventional manner to the peptide chemistry described in,
The desired compound represented by the general formula [I] can be easily produced with high yield.

The diazonium salt used in the present invention is preferably one which is not easily affected by other urinary components in the detection of leukocytes in urine, for example, 2-methoxy-4-
Morpholinobenzenediazonium-tetrachlorozincate, sodium 1-diazo-2-naphthol-4-sulfonate, 4-dimethylaminobenzenediazonium-tetrafluoroborate, 2,4-dimethoxybenzenediazonium
-Tetrafluoroborate, 4-methoxybenzenediazonium-tetrafluoroborate and the like can be mentioned.

The reagent composition for measurement according to the present invention comprises an ester degrading enzyme or a protein degrading enzyme in a biological sample, particularly urine,
It is particularly suitable for measuring esterases present in leukocytes, and by using this, leukocytes in urine can be detected rapidly and with high sensitivity in a deep color tone of purple to blue that is hardly affected by coexisting substances. . The measuring reagent composition according to the present invention can of course be used as a liquid reagent, but is most advantageous when used as a test piece. The concentration of the compound represented by the general formula [I] according to the present invention in the reagent composition for measurement is not particularly limited as long as it is an amount capable of detecting the target enzyme activity. When used in the form, the concentration in the impregnating solution used for holding the absorbent carrier is usually 0.1 to 100 mM, preferably 1 to 10 mM. The concentration of the diazonium salt used is not particularly limited, but when it is used in the form of a test piece, for example, the concentration in the impregnating liquid used for holding it on the absorbent carrier is usually 0.1 to 100 mM, preferably 1
~ 10 mM.

The type of buffer used in the present invention may be any as long as it maintains the pH at 6 to 10, preferably 7 to 9 and does not interfere with the detectable response reaction. For example, boric acid. Salt buffer, phosphate buffer, Tris
(Hydroxymethyl) aminomethane buffer, barbiturate buffer and the like can be mentioned, but of course the invention is not limited thereto. The concentration of these buffers used in the measuring reagent is not particularly limited as long as it can maintain the pH at 6 to 10, preferably 7 to 9, but is not limited thereto. The concentration in the buffer solution used for holding the measuring reagent on the absorptive carrier is usually preferably 0.1 to 1 M.

In the reagent for measuring the esterase or proteolytic enzyme according to the present invention, in addition to the compound represented by the general formula [I], a diazonium salt, a buffer, a reaction accelerator, a stabilizer, a wetting agent, etc. The additive may be included. The usage amount, usage concentration, etc. may be appropriately selected from the usage amount, concentration range, etc. usually used in this field. Examples of the reaction accelerator include 1-hexanol, 1-octanol, 1-decanol, 1-undecanol, 1-dodecanol, 6-chloro-1-hexanol, 2-chlorocyclohexanol,
6-bromo-1-hexanol, 8-bromo-1-octanol,
Alcohols such as 11-bromo-1-undecanol, 12-bromo-1-dodecanol, 1,10-decanediol, and 2-hexyloxyethanol are used as stabilizers, for example, trimorphoyl phosphate, and as humectants, for example,
Examples thereof include polyvinylpyrrolidone, polyethylene glycol, laurylpyridinium chloride, polyoxyethylene nonylphenyl ether, and the like, but are not limited thereto.

The test piece for detecting leukocytes according to the present invention contains a compound represented by the general formula [I], a diazonium salt and a buffering agent, and if necessary, reagents such as a reaction accelerator, a stabilizer and a wetting agent. A reagent composition comprising the above can be retained by impregnating an absorbent carrier by a conventional method and drying, or can be easily prepared by coating or coating the reagent composition on a film and retaining the same. obtain. The preparation method of the present invention will be described more concretely by taking the case of using an absorbent carrier as an example. That is, the compound represented by the general formula [I] and a diazonium salt, pH 6 to 1
A buffering agent capable of holding 0, preferably 7 to 9, and further necessary reagents such as a reaction accelerator, a stabilizer and a wetting agent,
One or two or more impregnating solutions prepared by dissolving in water or an alcohol such as methanol or ethanol, a polar solvent such as acetone or dimethylformamide (DMF), or a mixed solvent of these polar solvents and water as appropriate. And prepare
If a suitable absorbent carrier is dipped in the impregnating solution once or several times and dried, and then cut into a predetermined size if necessary,
The test piece of the present invention can be obtained. The test piece thus obtained may be used as it is, or may be used by adhering it to one end of a support such as a film piece of polyvinyl chloride, polystyrene or the like.

The absorptive carrier and film used for preparing the test piece of the present invention are not particularly limited as long as they are those usually used in the field of dry chemistry. Preferable examples of the carrier include filter paper, non-woven fabric, glass fiber and the like, and examples of the film include polystyrene sheet having a thickness of about 300 μm. When the test piece of the present invention is prepared using a film, it is necessary to use an appropriate film forming agent in order to form a thin film holding a necessary reagent. Examples of these film-forming agents include, but are not limited to, those commonly used in the field of dry chemistry, for example, polyvinyl alcohol resin, polyvinylpyrrolidone resin, polyacrylamide resin, polyethylene oxide resin, or Water-soluble synthetic resin such as polyvinyl acetate resin, water-soluble cellulose derivative such as methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose or carboxymethyl cellulose, starch, polysaccharide, gelatin, casein, gum arabic, or sodium alginate Examples include natural polymers. The measurement reagent composition according to the present invention may be used by holding it on a test piece and using it, for example, by adding and dissolving it directly in a sample for measurement or containing the measurement reagent. Since it is also possible to add a sample to the solution to perform the measurement, the measurement may be performed in this manner. less than,
The present invention will be described in more detail with reference to Reference Examples and Examples, but these do not limit the scope of the present invention.

[0017]

【Example】

Reference example 1. Synthesis of carboxymethyl thiobenzimidate hydrobromide To benzene solution 80 ml of 5.7 g of thiobenzamide (manufactured by Aldrich) was added 40 ml of benzene solution of 5.8 g of bromoacetic acid,
The reaction was carried out at room temperature for 15 hours. The precipitated crystals were collected by filtration, washed with ether and dried to obtain 9.0 g of carboxymethyl thiobenzimidate hydrobromide (yield 78.8%). Reference Example 2.2 Synthesis of 2-phenyl-4 (5H) thiazolone To 8.3 g of carboxymethyl thiobenzimidate hydrobromide obtained in Reference Example 1, 35 ml of pyridine was added and reacted at room temperature for 15 minutes. After completion of the reaction, 250 ml of cold water was added, and the precipitated crystals were collected by filtration and washed with cold water. Recrystallization from ethanol gave yellow crystals of 2-phenyl-4 (5H) thiazolone 3.5g.
Was obtained (yield 66.0%). Reference example 3. Synthesis of N-tosyl-L-alanyl chloride A mixture of 12 g of N-tosyl-L-alanine and 25 ml of thionyl chloride was reacted at 50 ° C for 90 minutes. After completion of the reaction, 200 ml of hexane was added under ice cooling for crystallization to obtain 11.6 g of N-tosyl-L-alanyl chloride (yield 90.0%). Melting point: 90-100
° C.

Example 1 Synthesis of 2-phenyl-4- (N-tosyl-L-alanyloxy) thiazole 3.0 g of 2-phenyl-4 (5H) thiazolone obtained in Reference Example 2,
30 ml of DMF solution of 2.5 g of N, N-dimethylaminopyridine
Under ice-cooling, 5.32 g of N-tosyl-L-alanyl chloride obtained in Reference Example 3 was added, and the mixture was reacted for 2 hours. After completion of the reaction, the solvent was distilled off, the residue was extracted with ethyl acetate, and the extract was washed with water. After drying the ethyl acetate layer with anhydrous sodium sulfate,
Ethyl acetate was distilled off, and the obtained residue was purified by silica gel column [4.6φ × 10 cm, packing material: Wakogel C-300 (trade name of Wako Pure Chemical Industries, Ltd.), eluent: chloroform],
4.09 g of white crystals of 2-phenyl-4- (N-tosyl-L-alanyloxy) thiazole were obtained (yield 60.0%). Melting point: 118-120 ° C. IR (KBr) cm ^-1 : 3280 (NH), 3110 (CH), 1760 (ester), 1600 (phenyl). ¹ H-NMR (270 MHz, CDCl ₃ ) δppm: 1.56 (d, 3H, CH-C
_{H 3, J = 7.02Hz),} 2.34 (s, 3H, Ar-C H 3), 4.30 (q, 1H, C H -CH 3,
J = 7.02,8.64Hz), 5.49 (d, 1H, NH, J = 8.64Hz), 6.90 (s, 1H,
Thiasol C-5H), 7.2-7.9 (9H, ArH). Elemental analysis value Calculated value as C ₁₉ H ₁₈ N ₂ O ₄ S ₂ (%): C 56.69, H 4.51, N 6.96 Measured value (%): C 56.66, H 4.43, N 6.95.

Reference Example 4. Synthesis of 4-methoxythiobenzamide To a solution of 13.3 g of 4-methoxybenzonitrile, 30 ml of pyridine and 14 ml of triethylamine, hydrogen sulfide gas was added at 3.5.
It was introduced for a time and reacted. After the reaction is completed, the reaction solution is treated with water 500m
It was poured into 1 and the precipitated crystal was collected by filtration. Recrystallized from toluene to give 4-methoxythiobenzamide as yellow needle crystals.
8.7 g was obtained (yield 52.1%). Melting point: 146-147 ° C. Reference example 5. Synthesis of carboxymethyl 4-methoxythiobenzimidate hydrobromide 3.5 g of 4-methoxythiobenzamide obtained in Reference Example 4 and 3.48 g of bromoacetic acid were reacted and post-treated in the same manner as in Reference Example 1, 5.22 g of carboxymethyl 4-methoxythiobenzimidate hydrobromide was obtained (yield 81.6%). Reference Example 6.2 Synthesis of 2- (4′-methoxy) phenyl-4 (5H) thiazolone To carboxymethyl 4-methoxythiobenzimidate hydrobromide (5.2 g) obtained in Reference Example 5 was added pyridine (35 ml),
The same treatment as in Reference Example 2 was carried out to obtain 3.0 g of yellow crystals of 2- (4'-methoxy) phenyl-4 (5H) thiazolone (yield 85.2).
%).

Example 2.2- (4'-methoxy) phenyl-4-
Synthesis of (N-tosyl-L-alanyloxy) thiazole 3.0-g of 2- (4'-methoxy) phenyl-4 (5H) thiazolone obtained in Reference Example 6 and 4.55 g of N-tosyl-L-alanyl chloride obtained in Reference Example 3 were carried out. After treating in the same manner as in Example 1, the crystals were crystallized from ethyl acetate-ether-n-hexane to give 2- (4 '
-Methoxy) phenyl-4- (N-tosyl-L-alanyloxy)
3.42 g of pale yellow crystals of thiazole were obtained (yield 54.9%). Melting point: 110-111 ° C. IR (KBr) cm ^-1 : 3300 (NH), 3140 (CH), 1775 (ester), 1610 (phenyl). ¹ H-NMR (270 MHz, CDCl ₃ ) δppm: 1.57 (d, 3H, CH—C H ₃ ,
J = 7.56Hz), 2.36 (s, 3H, Ar-C H ₃ ), 3.86 (s, 3H, Ar-OC H ₃ ),
4.27 (q, 1H, C H -CH ₃ , J = 7.56,9.18Hz), 5.28 (d, 1H, NH, J = 9.
18Hz), 6.81 (s, 1H, Thiasol C-5H), 6.8 to 7.8 (8H, ArH). Elemental analysis value Calculated value as C ₂₀ H ₂₀ N ₂ O ₅ S ₂ (%): C 55.54, H 4.66, N 6.48 Measured value (%): C 55.48, H 4.61, N 6.37.

Reference Example 7. Synthesis of 4-fluorothiobenzamide A solution consisting of 12.1 g of 4-fluorobenzonitrile, 30 ml of pyridine and 14 ml of triethylamine was treated in the same manner as in Reference Example 4 to give yellow scale of 4-fluorothiobenzamide. 13.9 g of crystalline crystals was obtained (yield 89.7%). Melting point: 144-145 ° C. Reference example 8. Synthesis of carboxymethyl 4-fluorothiobenzimidate hydrobromide 3.88 g of 4-fluorothiobenzamide obtained in Reference Example 7 and 4.17 g of bromoacetic acid were reacted and post-treated in the same manner as in Reference Example 1, 5.45 g of carboxymethyl 4-fluorothiobenzimidate hydrobromide was obtained (yield 74.2%). Reference Example 9.2 Synthesis of 2- (4'-fluoro) phenyl-4 (5H) thiazolone To 5.4 g of carboxymethyl 4-fluorothiobenzimidate hydrobromide obtained in Reference Example 35, 35 ml of pyridine was added,
The same treatment as in Reference Example 2 was carried out to obtain 1.91 g of yellow crystals of 2- (4′-fluoro) phenyl-4 (5H) thiazolone (yield 53.
Five%).

Example 3.2 2- (4'-Fluoro) phenyl-4-
Synthesis of (N-tosyl-L-alanyloxy) thiazole 2.9 g of 2- (4'-fluoro) phenyl-4 (5H) thiazolone obtained in Reference Example 9 and 3.06 g of N-tosyl-L-alanyl chloride obtained in Reference Example 3 were carried out. After processing in the same manner as in Example 1,
Crystallized from ether to give 2- (4'-fluoro) phenyl-4-
White crystals of (N-tosyl-L-alanyloxy) thiazole
2.21 g was obtained (yield 54.0%). Melting point: 121-122 ° C. IR (KBr) cm ⁻¹ : 3310 (NH), 3140 (CH), 1780 (ester), 1600 (phenyl). ¹ H over NMR (270MHz, CDCl3) δppm: 1.57 (d, 3H, CH-C H 3,
J = 7.29Hz), 2.36 (s, 3H, Ar-C H ₃ ), 4.29 (q, 1H, C H -CH ₃ , J =
7.29,9.18Hz), 5.38 (d, 1H, NH, J = 9.18Hz), 6.89 (s, 1H, thiazol C-5H), 7.1-7.9 (8H, ArH). Elemental analysis value Calculated value as C ₁₉ H ₁₇ FN ₂ O ₄ S ₂ (%): C 54.24, H 4.08, N 6.66 Measured value (%): C 54.05, H 4.15, N 6.65.

Reference Example 10. Synthesis of 3-fluorothiobenzamide A solution of 12.1 g of 3-fluorobenzonitrile, 30 ml of pyridine and 14 ml of triethylamine was treated in the same manner as in Reference Example 4 to give yellow scales of 3-fluorothiobenzamide. 12.8 g of crystalline crystals was obtained (yield 82.6%). Melting point: 104-105 ° C. Reference example 11. Synthesis of carboxymethyl 3-fluorothiobenzimidate hydrobromide 3.88 g of 3-fluorothiobenzamide obtained in Reference Example 10 and 4.17 g of bromoacetic acid were reacted and post-treated in the same manner as in Reference Example 1, 6.34 g of carboxymethyl 3-fluorothiobenzimidate hydrobromide was obtained (yield 86.2%). Reference Example 12. Synthesis of 2- (3'-fluoro) phenyl-4 (5H) thiazolone To carboxymethyl 3-fluorothiobenzimidate hydrobromide (6.3 g) obtained in Reference Example 11, 35 ml of pyridine was added, and Reference Example 2 2- (3'-fluoro)
2.21 g of yellow crystals of phenyl-4 (5H) thiazolone were obtained (yield 52.9%).

Example 4.2 2- (3'-Fluoro) phenyl-4-
Synthesis of (N-tosyl-L-alanyloxy) thiazole 2.2-g of 2- (3'-fluoro) phenyl-4 (5H) thiazolone obtained in Reference Example 12 and 3.53 g of N-tosyl-L-alanyl chloride obtained in Reference Example 3 were carried out. After treating in the same manner as in Example 1, crystallization from ether gave 1.70 g of white crystals of 2- (3'-fluoro) phenyl-4- (N-tosyl-L-alanyloxy) thiazole (yield 33.9%). Melting point: 100-101 ° C. IR (KBr) cm ⁻¹ : 3320 (NH), 3130 (CH), 1780 (ester), 1610 (phenyl). ¹ H-NMR (270 MHz, CDCl ₃ ) δppm: 1.58 (d, 3H, CH—C H ₃ ,
J = 7.29Hz), 2.37 (s, 3H, Ar-C H ₃ ), 4.30 (q, 1H, C H -CH ₃ , J =
7.29,9.18Hz), 5.29 (d, 1H, NH, J = 9.18Hz), 6.94 (s, 1H, thiazol C-5H), 6.9 to 7.8 (8H, ArH). Elemental analysis value Calculated value as C ₁₉ H ₁₇ FN ₂ O ₄ S ₂ (%): C 54.27, H 4.08, N 6.66 Actual value (%): C 54.05, H 4.19, N 6.65.

Reference Example 13. Synthesis of 2-fluorothiobenzamide A solution consisting of 12.1 g of 2-fluorobenzonitrile, 30 ml of pyridine and 14 ml of triethylamine was treated in the same manner as in Reference Example 4 to give a yellow needle of 2-fluorothiobenzamide. 10.2 g of crystalline crystals was obtained (yield 65.8%). Melting point: 80.5-81 ° C. Reference example 14. Synthesis of carboxymethyl 2-fluorothiobenzimidate hydrobromide 2.88 g of 2-fluorothiobenzamide obtained in Reference Example 13 and 4.17 g of bromoacetic acid were reacted and post-treated in the same manner as in Reference Example 1, 7.00 g of carboxymethyl 2-fluorothiobenzimidate hydrobromide was obtained (yield 95.0%). Reference Example 15. Synthesis of 2- (2'-fluoro) phenyl-4 (5H) thiazolone To carboxymethyl 2-fluorothiobenzimidate hydrobromide 7.0 g obtained in Reference Example 14, pyridine 35 ml was added, and Reference Example 2 2- (2'-fluoro)
4.03 g of yellow crystals of phenyl-4 (5H) thiazolone were obtained (yield 83.3%).

Example 5.2 2- (2'-Fluoro) phenyl-4-
Synthesis of (N-tosyl-L-alanyloxy) thiazole 2- (2'-fluoro) phenyl-4 (5H) thiazolone 2.34 g obtained in Reference Example 15 and N-tosyl-L-alanyl chloride 3.77 g obtained in Reference Example 3 were carried out. The mixture was treated in the same manner as in Example 1 and then crystallized from ether to obtain 2.77 g of white crystals of 2- (2'-fluoro) phenyl-4- (N-tosyl-L-alanyloxy) thiazole (yield 55.0%). Melting point: 109-110 ° C. IR (KBr) cm ^-1 : 3290 (NH), 3110 (CH), 1765 (ester), 1600 (phenyl). ¹ H-NMR (270 MHz, CDCl ₃ ) δppm: 1.58 (d, 3H, CH—C H ₃ ,
J = 6.75Hz), 2.36 (s, 3H, Ar-C H ₃ ), 4.30 (q, 1H, C H -CH ₃ , J =
6.75,9.18Hz), 5.33 (d, 1H, NH, J = 9.18Hz), 7.00 (s, 1H, thiazol C-5H), 7.15 to 8.2 (8H, ArH). Elemental analysis value Calculated value as C ₁₉ H ₁₇ FN ₂ O ₄ S ₂ (%): C 54.27, H 4.08, N 6.66 Measured value (%): C 54.01, H 4.23, N 6.65.

Reference Example 16. Synthesis of 3,5-dimethoxythiobenzamide A solution of 16.5-g 3,5-dimethoxybenzonitrile, 40 ml pyridine and 14 ml triethylamine was treated in the same manner as in Reference Example 4 to obtain a crude product. The crystals were recrystallized from ethanol to obtain 14.4 g of yellow needle crystals of 3,5-dimethoxythiobenzamide (yield 73.1%). Melting point: 117-118 ° C. Reference Example 17. Synthesis of carboxymethyl 3,5-dimethoxythiobenzimidate hydrobromide 3,5-dimethoxythiobenzamide 4.93 obtained in Reference Example 16
g and 4.17 g of bromoacetic acid in the same manner as in Reference Example 1,
Post-treatment gave 7.30 g of carboxymethyl 3,5-dimethoxythiobenzimidate hydrobromide (yield 86.8
%). Reference Example 18. Synthesis of 2- (3 ', 5'-dimethoxy) phenyl-4 (5H) thiazolone Carboxymethyl 3,5-dimethoxythiobenzimidate hydrobromide 7.3 g obtained in Reference Example 17 and pyridine 35 ml Was added and treated in the same manner as in Reference Example 2 to obtain 4.56 g of yellow crystals of 2- (3 ′, 5′-dimethoxy) phenyl-4 (5H) thiazolone (yield 88.5%).

Example 6. Synthesis of 2- (3 ', 5'-dimethoxy) phenyl-4- (N-tosyl-L-alanyloxy) thiazole 2- (3', 5'-dimethoxy) phenyl-4 (5H )
2.85 g of thiazolone and 3.77 g of N-tosyl-L-alanyl chloride obtained in Reference Example 3 were treated in the same manner as in Example 1 and then crystallized from ether to give 2- (3 ', 5'-dimethoxy). )
2.12 g of white crystals of phenyl-4- (N-tosyl-L-alanyloxy) thiazole were obtained (yield 38.2%). Melting point: 110-112 ° C. IR (KBr) cm ^-1 : 3280 (NH), 3110 (CH), 1760 (ester), 1600 (phenyl). ¹ H-NMR (270 MHz, CDCl ₃ ) δppm: 1.58 (d, 3H, CH—C H ₃ ,
J = 7.02Hz), 2.36 (s, 3H, Ar-C H ₃ ), 3.85 (s, 6H, 2 × OC H ₃ ),
4.29 (q, 1H, C H -CH ₃ , J = 7.02,8.91Hz), 5.28 (d, 1H, NH, J = 8.
91Hz), 6.90 (s, 1H, Thiasol C-5H), 7.0 to 7.8 (7H, ArH). Elemental analysis value Calculated value as C ₂₁ H ₂₂ N ₂ O ₆ S ₂ (%): C 54.53, H 4.79, N 6.06 Actual value (%): C 54.22, H 4.99, N 5.98.

Reference Example 1 Synthesis of 4-methylthiobenzamide A solution of 11.7 g of 4-methylbenzonitrile, 30 ml of pyridine and 14 ml of triethylamine was treated in the same manner as in Reference Example 4 to give 4-methylthiobenzamide yellow needles. crystal
9.2 g was obtained (yield 60.9%). Melting point: 165-166 ° C. Reference example 20. Synthesis of carboxymethyl 4-methylthiobenzimidate hydrobromide 3.78 g of 4-methylthiobenzamide obtained in Reference Example 19 and 4.17 g of bromoacetic acid were reacted and post-treated in the same manner as in Reference Example 1 to give carboxymethyl. 5.52 g of 4-methylthiobenzimidate hydrobromide was obtained (yield 76.1%). Reference Example 2 Synthesis of 2- (4′-methyl) phenyl-4 (5H) thiazolone To 5.5 g of carboxymethyl 4-methylthiobenzimidate hydrobromide obtained in Reference Example 20, 35 ml of pyridine was added,
The same treatment as in Reference Example 2 was carried out to obtain 2.21 g of 2- (4-methyl) phenyl-4 (5H) thiazolone yellow crystals (yield 60.9).
%).

Example 7 Synthesis of 2- (4'-methyl) phenyl-4- (N-tosyl-L-alanyloxy) thiazole 2.2-g of 2- (4'-methyl) phenyl-4 (5H) thiazolone obtained in Reference Example 21 After treating 3.61 g of N-tosyl-L-alanyl chloride obtained in Example 3 in the same manner as in Example 1,
Crystallized from ether to give 2- (4'-methyl) phenyl-4-
White crystals of (N-tosyl-L-alanyloxy) thiazole
4.35 g was obtained (yield 90.8%). Melting point: 143-144 ° C. IR (KBr) cm ^-1 : 3290 (NH), 3110 (CH), 1760 (ester), 1600 (phenyl). ¹ H-NMR (270 MHz, CDCl ₃ ) δppm: 1.57 (d, 3H, CH—C H ₃ ,
J = 7.02Hz), 2.35 (s, 3H, Ar-C H ₃ (tosyl)), 2.40 (s, 3H, Ar-C H
₃ ), 4.28 (q, 1H, C H -CH ₃ , J = 7.02,8.64Hz), 5.31 (d, 1H, NH,
J = 8.64Hz), 6.85 (s, 1H, Thiasol C-5H), 7.2 ~ 7.8 (8H, Ar
H). Elemental analysis _{C 20 H 20 N 2 O 4} S 2 Calculated (%): C 57.67, H 4.84, N 6.73 Found (%): C 57.79, H 4.78, N 6.65.

Reference Example 2 Synthesis of 2-thionaphthamide A solution of 9.75 g of 2-naphthonitrile, 20 ml of pyridine and 9.2 ml of triethylamine was treated in the same manner as in Reference Example 4 to give red-brown needle crystals of 2-thionaphthamide. 8.24 g was obtained (yield 69.2%). Melting point 143-144 ° C. Reference Example 23. Synthesis of carboxymethyl 2-thionaphthimidate hydrobromide 5.0 g of 2-thionaphthamide obtained in Reference Example 22 and 80 benzene
30 ml of a benzene solution of 3.7 g of bromoacetic acid was added to a mixed solution of 40 ml of THF and 40 ml of THF, and the reaction and post-treatment were carried out in the same manner as in Reference Example 1 to obtain carboxymethyl 2-thionaphthoimidate hydrobromide 7.2.
6 g was obtained (yield 83.2%). Reference Example 24.2 Synthesis of 2- (2naphthyl) -4 (5H) thiazolone 35 ml of pyridine was added to 7.26 g of carboxymethyl 2-thionaphthimidate hydrobromide obtained in Reference Example 23, and the same as in Reference Example 2. By the method described above, 3.78 g of yellow crystals of 2- (2naphthyl) -4 (5H) thiazolone were obtained (yield 75.6%).

Example 8.2 Synthesis of 2- (2naphthyl) -4- (N-tosyl-L-alanyloxy) thiazole Obtained in Reference Example 24.
3.5 g of 2- (2 naphthyl) -4 (5H) thiazolone and 4.9 g of N-tosyl-L-alanyl chloride obtained in Reference Example 3 were treated in the same manner as in Example 1, and then treated with chloroform-hexane. Recrystallization was performed to obtain 3.20 g of white crystals of 2- (2naphthyl) -4- (N-tosyl-L-alanyloxy) thiazole (yield 51.7).
%). Melting point: 137-138 ° C. IR (KBr) cm ⁻¹ : 3275 (NH), 3150 (CH), 1780 (ester), 1610 (phenyl) ¹ H-NMR (270 MHz, CDCl ₃ ) δppm: 1.59 (d, 3H, CH—CH ₃ ,
J = 7.29Hz), 2.36 (s , 3H, Ar-CH 3), 4.25~4.40 (m, 1H, C H -C
H ₃ ), 5.27 (d, 1H, NH, J = 8.64Hz), 6.94 (s, 1H, thiazol C-5
H), 7.2-8.4 (11H, ArH). Elemental analysis _{C 23 H 20 N 2 O 4} S 2 Calculated (%): C 61.04, H 4.45, N 6.19 Found (%): C 60.95, H 4.38, N 6.18.

Reference Example 25.2 Synthesis of 2-phenyl-3,5-dihydroimidazol-4-one Benzamidine hydrochloride 3 g, glycine ethyl ester hydrochloride 8.4 g, sodium hydrogencarbonate 8 g and methanol 70 ml were mixed in a nitrogen stream at 2.5 The mixture was refluxed for a period of time. After cooling, chloroform and water were added for extraction, the chloroform layer was washed with saturated saline and then dried over anhydrous sodium sulfate. Chloroform was distilled off under reduced pressure to give 2-phenyl-
700 mg of red crystals of 3,5-dihydroimidazol-4-one were obtained.

Example 9 Synthesis of 2-phenyl-4- (N-tosyl-L-alanyloxy) imidazole 500 mg of 2-phenyl-3,5-dihydroimidazol-4-one obtained in Reference Example 25, 0.6 ml of trifluoroacetic acid and pyridine
To a solution of 0.3 ml of tetrahydrofuran dissolved in 10 ml of tetrahydrofuran, a solution of 1.0 g of N-tosyl-L-alanyl chloride obtained in Reference Example 3 in 4 ml of tetrahydrofuran was added under cooling to -20 to -10 ° C over 10 minutes. After reacting at the same temperature for 1 hour, 25 ml of 0.4 M citric acid aqueous solution was added and the mixture was extracted with ethyl acetate. The ethyl acetate layer was washed successively with aqueous sodium hydrogen carbonate solution and saturated brine and dried over anhydrous sodium sulfate, and ethyl acetate was evaporated under reduced pressure. The obtained residue is a silica gel column [filler: Wakogel FC-40 (trade name of Wako Pure Chemical Industries, Ltd.), eluent: 5
% Acetone-Chloroform] and then 2-phenyl-4-
339 mg of slightly yellow crystals of (N-tosyl-L-alanyloxy) imidazole were obtained (yield 31%). Melting point: 25-25 ° C. IR (KBr) cm ⁻¹ : 3300 (NH), 1760 (ester), 1670 (C
= N), 1600 (phenyl). ¹ H-NMR (270 MHz, CDCl ₃ ) δppm: 1.35 (d, 3H, CH—C H ₃ ,
J = 7.02Hz), 2.34 (s, 3H, Ar-C H ₃ ), 3.80 ~ 4.00 (m, 1H, C H -C
H ₃ ), 5.55 (d, 1H, NH, J = 8.64Hz), 6.95 (s, 1H, Imidazole C-5
H), 7.2-7.9 (9H, ArH). Elemental analysis value Calculated value (%) as C ₁₉ H ₁₈ N ₃ O ₄ S: C 59.36, H 4.72, N 10.93 Measured value (%): C 59.24, H 4.85, N 10.90.

Example 10 [Preparation of test piece] A filter paper was immersed in the following impregnating solution 1 and dried, and then the following impregnating solution containing 2-phenyl-4- (N-tosyl-L-alanyloxy) thiazole obtained in Example 1 was used. It was dipped in 2 and dried to prepare a test piece. (Impregnation liquid 1) Boric acid 4.94 g Sodium hydroxide 0.40 g Total amount of distilled water 100 ml (pH 7.0) (Impregnation liquid 2) 2-phenyl-4- (N-tosyl-L-alanyloxy) thiazole 121 mg 2-methoxy- 4-morpholinobenzenediazonium-tetrachlorozincate 97.2 mg Methanol solution containing reaction accelerator 100 ml (6-bromo-1-hexanol 3% (W /
V), 11-bromo-1-undecanol 1% (W / V), or 12
-Bromo-1-dodecanol 1% (W / V) is used) [Operation and Results] Immediately after immersing each of the above-mentioned test pieces in urine to which a white blood cell fraction isolated from human whole blood was added,
Upon visual observation, the test piece was colored blue-violet according to its concentration. Further, it was possible to detect white blood cells with a low concentration of 10 cells / μl 2 minutes after the immersion in the sample. Table 1 shows the results of measuring the reflectance at a measurement wavelength of 565 nm (reference wavelength of 760 nm) using Pretester RM-405 (manufactured by Wako Pure Chemical Industries, Ltd.).

[Table 1]

Example 11 [Preparation of test piece] 2-phenyl in impregnating solution 2 of Example 10
Except that 126 mg of 2- (2'-fluoro) phenyl-4- (N-tosyl-L-alanyloxy) thiazole obtained in Example 5 was used instead of -4- (N-tosyl-L-alanyloxy) thiazole. Using the same reagent as in Example 10, a test piece was prepared by the same operation. [Measurement and Results] Each test piece was immediately pulled up after being immersed in urine to which the white blood cell fraction isolated from human whole blood was added, and visually observed, and the test piece was colored blue according to its concentration.

Example 12 [Preparation of test piece] 2-phenyl in impregnating solution 2 of Example 10
Except that 126 mg of 2- (3'-fluoro) phenyl-4- (N-tosyl-L-alanyloxy) thiazole obtained in Example 4 was used instead of -4- (N-tosyl-L-alanyloxy) thiazole. Using the same reagent as in Example 10, a test piece was prepared by the same operation. [Measurement and Results] Each test piece was immediately pulled up after being immersed in urine to which the white blood cell fraction isolated from human whole blood was added, and visually observed, and the test piece was colored blue according to its concentration.

Example 13 [Preparation of test piece] 2-phenyl in impregnating solution 2 of Example 10
Except that 126 mg of 2- (4'-fluoro) phenyl-4- (N-tosyl-L-alanyloxy) thiazole obtained in Example 3 was used instead of -4- (N-tosyl-L-alanyloxy) thiazole. Using the same reagent as in Example 10, a test piece was prepared by the same operation. [Measurement and Results] Each test piece was immediately pulled up after being immersed in urine containing a white blood cell fraction isolated from human whole blood and visually observed, and the test piece exhibited a mauve color depending on its concentration. .

Example 14 [Preparation of test piece] 2-phenyl in impregnating solution 2 of Example 10
-4- (N-tosyl-L-alanyloxy) thiazole was replaced by 2- (4'-methyl) phenyl-4- (N-tosyl) obtained in Example 7.
Using the same reagents as in Example 10 except that 125 mg of -L-alanyloxy) thiazole was used, test pieces were prepared by the same procedure. [Measurement and Results] Each test piece was immediately pulled up after being immersed in urine containing a white blood cell fraction isolated from human whole blood and visually observed, and the test piece exhibited a mauve color depending on its concentration. .

Example 15 [Preparation of test piece] 2-phenyl in impregnating solution 2 of Example 10
Except that 129 mg of 2- (4'-methoxy) phenyl-4- (N-tosyl-L-alanyloxy) thiazole obtained in Example 2 was used instead of -4- (N-tosyl-L-alanyloxy) thiazole. Using the same reagent as in Example 10, a test piece was prepared by the same operation. [Measurement and Results] Each test piece was immediately pulled up after being immersed in urine containing a white blood cell fraction isolated from human whole blood and visually observed, and the test piece exhibited a mauve color depending on its concentration. .

Example 16 [Preparation of test piece] 2-phenyl in impregnating solution 2 of Example 10
-4- (N-tosyl-L-alanyloxy) thiazole was replaced by 2- (3 ', 5'-dimethoxy) phenyl-4- (N
A test piece was prepared by the same procedure as in Example 10 except that 139 mg of -tosyl-L-alanyloxy) thiazole was used. [Measurement and Results] Each test piece was immediately pulled up after being immersed in urine to which the white blood cell fraction isolated from human whole blood was added, and visually observed, and the test piece was colored blue according to its concentration.

Example 17 [Preparation of test piece] 2-phenyl in impregnating solution 2 of Example 10
Example 1 except that 136 mg of 2- (2naphthyl) -4- (N-tosyl-L-alanyloxy) thiazole obtained in Example 8 was used instead of -4- (N-tosyl-L-alanyloxy) thiazole.
Using the same reagent as 0, a test piece was prepared by the same operation. [Measurement and Results] Each test piece was immediately pulled up after being immersed in urine to which the white blood cell fraction isolated from human whole blood was added, and visually observed, and the test piece was colored blue according to its concentration.

Example 18 [Preparation of test piece] A filter paper was immersed in the following impregnating solution 1 and dried, and then the following impregnating solution containing 2-phenyl-4- (N-tosyl-L-alanyloxy) thiazole obtained in Example 1 was used. It was dipped in 2 and dried to prepare a test piece. (Impregnation liquid 1) Boric acid 2.47 g Sodium hydroxide 0.36 g Distilled water Total amount 100 ml (pH 8.0) (Impregnation liquid 2) 2-phenyl-4- (N-tosyl-L-alanyloxy) thiazole 121 mg 1-diazo 2 -Sodium naphthol-4-sulfonate 81.7 mg 1-decanol 1.0 g Methanol 100 ml [Measurement and results] The above test piece was immediately pulled up after immersion in urine containing leukocyte fraction isolated from human whole blood and visually observed. However, the test piece was colored blue according to its density.

Example 19 [Preparation of test piece] 1-diazo-2 in impregnating solution 2 of Example 18
A test piece was prepared in the same manner as in Example 18 except that 97.2 mg of 2-methoxy-4-morpholinobenzenediazonium-tetrachlorozincate was used instead of sodium naphthol-4-sulfonate. [Measurement and Results] The test piece was immediately immersed in urine containing 300 mg / dl of hemoglobin without leukocytes and 300 mg / dl of leukocytes at 300 mg / dl of leukocytes, and immediately pulled up and visually observed 2 minutes later. However, the former was light brown and the latter was light dark purple, and the two were easy to distinguish.

Comparative Example 1 [Preparation of test piece] 2-phenyl in impregnating solution 2 of Example 18
The same reagents as in Example 18 were used, except that 115 mg of 3- (N-tosyl-L-alanyloxy) -5-phenylpyrrole was used instead of -4- (N-tosyl-L-alanyloxy) thiazole.
A test piece was prepared by the same operation. [Measurement and Results] The test piece was immediately immersed in urine containing 300 mg / dl of hemoglobin without leukocytes and 300 mg / dl of leukocytes at 300 mg / dl of leukocytes, and immediately pulled up and visually observed 2 minutes later. However, it was difficult to distinguish between the former and the latter because they were colored light brown.

Comparative Example 2. [Preparation of test piece] 2-phenyl in impregnating solution 2 of Example 18
108 mg of 3- (N-tosyl-L-alanyloxy) indole instead of 4- (N-tosyl-L-alanyloxy) thiazole
Example 18 except that 97.2 mg of 2-methoxy-4-morpholinobenzenediazonium-tetrachlorozincate was used in place of sodium 1-diazo-2-naphthol-4-sulfonate.
Using the same reagent as above, a test piece was prepared by the same operation. [Measurement and Results] The test piece was immediately immersed in urine containing 300 mg / dl of hemoglobin without leukocytes and 300 mg / dl of leukocytes at 300 mg / dl of leukocytes, and immediately pulled up and visually observed 2 minutes later. However, it was difficult to distinguish between the former and the latter because they were colored light brown.

Example 20 [Preparation of test piece] 2-phenyl in impregnating solution 2 of Example 18
Example 18 except that 116 mg of 2-phenyl-4- (N-tosyl-L-alanyloxy) imidazole obtained in Example 9 was used instead of -4- (N-tosyl-L-alanyloxy) thiazole.
Using the same reagent as above, a test piece was prepared by the same operation. [Measurement and Results] The test piece was immediately immersed in urine containing a white blood cell fraction isolated from human whole blood and then pulled up and visually observed. The test piece was colored purple according to its concentration.

Example 21 [Preparation of test piece] A filter paper was immersed in the following impregnating solution 1 and dried, and then the following impregnating solution containing 2-phenyl-4- (N-tosyl-L-alanyloxy) thiazole obtained in Example 1 was used. It was dipped in 2 and dried to prepare a test piece. (Impregnation liquid 1) Boric acid 4.94 g Sodium hydroxide 0.80 g Sodium chloride 1.46 g Polyvinylpyrrolidone (K-30, Wako Pure Chemical Industries, Ltd.) 2.0 g Distilled water 100 ml total (pH 8.0) (Impregnating liquid 2) 2-phenyl-4- (N-tosyl-L-alanyloxy) thiazole 80.5 mg 2-methoxy-4-morpholinobenzenediazonium-tetrachlorozincate 64.8 mg 1-decanol 1.0 g methanol 100 ml [Measurement and Results] The above test piece was immediately immersed in physiological saline containing the white blood cell fraction isolated from human whole blood and immediately pulled up, and 2 minutes later, using a pretester RM-405, at a measurement wavelength of 565 nm (reference wavelength of 760 nm). The reflectance was measured.
The results are shown in Table 2.

Comparative Example 3 [Preparation of test piece] 2-phenyl in impregnating solution 2 of Example 21
-4- (N-tosyl-L-alanyloxy) thiazole instead of 3- (N-tosyl-L-alanyloxy) indole 71.7mg
Using the same reagents as in Example 21 except that was used, a test piece was prepared by the same operation. [Measurement and Results] Immediately after immersing the above test piece in physiological saline to which a white blood cell fraction isolated from human whole blood was added, the test piece was pulled up, and 2 minutes later, using a pretester RM-405, a measurement wavelength 565 nm (reference wavelength 760 nm ) Was measured.
The results are also shown in Table 2.

[Table 2] Table 2 shows that the method using 2-phenyl-4- (N-tosyl-L-alanyloxy) thiazole of the present invention as a substrate is better than the conventional method using 3- (N-tosyl-L-alanyloxy) indole. It can be seen that a wide range of white blood cell concentrations can be measured with high sensitivity.

[0050]

INDUSTRIAL APPLICABILITY As described above, the present invention provides a novel amino acid ester and peptide ester that can be easily and inexpensively produced, a method for measuring an esterase or a proteinase using the compound, and a reagent composition for measurement. By using a test piece in which the measuring reagent according to the present invention is held on a carrier, leukocytes are rapidly and highly sensitive in a deep color tone (purple to blue) that is not easily affected by coexisting substances. It is an invention that has a remarkable effect in that it can be detected in the above, and is an extremely large invention that contributes to the related art.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location // C07K 5/04 8318-4H 7/06

Claims (4)

[Claims] 1. A compound represented by the general formula [I]: [In the formula, A is an amino acid residue whose amino group is protected or N
The terminal amino group represents a protected peptide residue, R represents an aryl group which may have a substituent, and X represents an oxygen atom, a sulfur atom or an imino group which may have a substituent. . ] The compound shown by these is used as a substrate, The measuring method of esterolytic enzyme or proteolytic enzyme. 2. A compound represented by the general formula [I]: [In the formula, A is an amino acid residue whose amino group is protected or N
The terminal amino group represents a protected peptide residue, R represents an aryl group which may have a substituent, and X represents an oxygen atom, a sulfur atom or an imino group which may have a substituent. . ] A reagent composition for measuring an esterolytic enzyme or a proteolytic enzyme, which comprises a compound represented by the formula [1], a diazonium salt and a buffer substance. 3. A test piece for detecting leukocytes, comprising the reagent composition according to claim 2 held on an absorptive carrier or film. 4. A compound represented by the general formula [II]: [In the formula, R'represents an aryl group, an alkoxy-substituted aryl group or a halogen-substituted aryl group, and X'represents a sulfur atom or an imino group. ] The compound shown by these.