JPH07103134B2 - Phosphate ester - Google Patents

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention relates to a novel phosphoric acid monoester. More specifically, it relates to a novel mono-4-arylazoaryl phosphate ester which is hydrolyzed by the action of alkaline phosphatase to release an azo dye.

2. Description of the Related Art It is extremely important for clinical examination to know the activity of alkaline phosphatase in human body fluid. That is, it is well known that when the activity of alkaline phosphatase is generally high, the metabolism of the liver, bone and the like is disordered and symptoms such as liver disease, rickets, osteosarcoma and thyroid abnormalities appear. Therefore, it is quite natural that various methods have been devised for measuring the activity of alkaline phosphatase. The alkaline phosphatase quantification method in daily clinical tests requires simple operation and good reproducibility. For this purpose, there are so-called colorimetric method and fluorescent method. That is,
The colorimetric method uses a compound that is hydrolyzed by the action of alkaline phosphatase to release a dye or a precursor of a dye as a substrate for an enzymatic reaction, and colorimetrically quantifies the dye released as a result of the enzymatic reaction, or This is a method to know the activity of alkaline phosphatase by colorimetrically determining the dye precursor that is released as a result of enzymatic reaction by chemically reacting it with a reagent and colorimetrically determining it. It has the advantage that it can be quantified using a colorimeter or a spectrophotometer. On the other hand, the fluorescent method uses a compound that is hydrolyzed by the action of alkaline phosphatase and releases a fluorescent substance as a substrate for an enzymatic reaction, and measures the fluorescent intensity of the fluorescent substance released as a result of the enzymatic reaction. It is a method of knowing the activity of alkaline phosphatase by doing so, and it has higher sensitivity than the colorimetric method, but it is susceptible to interference by a small amount of coexisting fluorescent substance and that the fluorometer is not widely used. There are difficulties. The present invention relates to a novel compound, a mono-4-arylazoaryl phosphate ester, which is a suitable substrate in a colorimetric assay for alkaline phosphatase. Conventionally, the following method has been used as a method for quantifying alkaline phosphatase by a colorimetric method. (1) This is a method for colorimetrically quantifying p-nitrophenol released as a result of hydrolysis by the action of alkaline phosphatase using p-nitrophenyl phosphate disodium as a substrate. A. Be
sey, O.S. H. Lowry, and M.M. J. Bro
ck, Journal of Biological
Chemistry, Volume 164, p. 321 (19
Issued in 1946). (2) A method for colorimetrically quantifying phenolphthalein released as a result of hydrolysis by the action of alkaline phosphatase using phenolphthalein phosphate as a substrate. L. Babson, S .; J.
Greeney, C.I. M. Coleman, and G.G.
E. Phillips, Clinical Chemi
It is described in the magazine, p. 482, published in 1966 (published in 1966). (3) Using thymolphthalein phosphate as a substrate,
It is a method for colorimetrically determining thymolphthalein released as a result of hydrolysis by the action of alkaline phosphatase. M. Colemann Clinic
al Chemistry, vol. 13, p. 401 (1
966). (4) A method for colorimetrically determining thymol blue released as a result of hydrolysis by the action of alkaline phosphatase using thymol blue monophosphate as a substrate, and a specific example thereof is described in JP-A-51-136662. There is. (5) Using phenyl phosphate as a substrate, the phenol released as a result of hydrolysis by the action of alkaline phosphatase is oxidatively condensed with 4-aminoantipyrine in the presence of red blood salt, and the resulting red quinone is colorimetrically determined. As a specific example, the method described in P. R. N. Kind, and E. J. King, Clinical Patholo
gy, Vol. 7, p. 322 (published in 1954); Kensei Watanabe et al., "Clinical Pathology", vol. 15, p. 708 (19).
1987) Toshimasa Nakayama et al., "Clinical Pathology", Vol. 23 (Summary of General Assembly), page 85 (issued in 1975). Among the conventional methods described above, the method (1) is most widely used as a method for quantifying alkaline phosphatase, but since the colorimetric wavelength is 410 nm, it is free from interference by colored substances such as bilirubin and hemoglobin in serum. However, there was a drawback that the sample blinding was required and the operation was complicated accordingly. In the conventional methods (2) to (5), since the colorimetric wavelength is in the long wave region, this drawback has been overcome. However, it is difficult to synthesize a highly pure substrate by the methods (2), (3) and (4) using a monophosphate ester of phthaleins as a substrate. In other words, these monophosphates are synthesized by hydrolyzing the corresponding monophthalodichloridate of phthalein obtained by reacting the corresponding phthalein with one hydroxyl group in the molecule with phosphorus oxychloride. If the amount of phthalein is large, the two hydroxyl groups in the phthalein molecule will be phosphorylated, and if the amount of phosphorus oxychloride to phthalein is small, 2 or 3 molecules of phthalein will react with 1 molecule of phosphorus oxychloride. Two or three kinds of things are generated. In any case, a compound unsuitable as a substrate for quantifying alkaline phosphatase is by-produced. Therefore, isolation and purification after the reaction became complicated, and it was difficult to obtain many highly pure products. On the other hand, in the method of the conventional method (5), it was necessary to go through a two-step reaction process of an enzyme reaction for liberating phenol and a color reaction, and the operation was so complicated. Therefore, the advent of an alkaline phosphatase assay method that does not have the above-mentioned drawbacks has been desired.

Therefore, the object of the present invention is to have excellent operability during measurement, to be less susceptible to interference by colored substances such as dyes in blood, to have excellent detection sensitivity, and to be used for synthesis and purification. It is an object of the present invention to provide a novel compound which is useful as a substrate for quantifying alkaline phosphatase without difficulty.

The above-mentioned object is to provide a novel mono-4- represented by the following general formula (I) or (II).
It has been found that it can be effectively achieved with arylazoaryl phosphates.

[Chemical 2] (In the formula, M is a hydrogen atom, a sodium atom or a potassium atom, one of R ¹ and R ² is a methylsulfonyl group,
The other of R ¹ and R ² is a nitro group, R ³ is a chlorine atom or a hydrogen atom, R ⁴ is a hydrogen atom, a hydroxyl group, a methyl group or a methylcarbonylamino group, and R ⁵ is a hydrogen atom or a chlorine atom. And R ⁶ is a methyl group or 3
A sulfamoylphenyl group. ) Each of the compounds represented by the general formula (I) or (II) is hydrolyzed by the action of alkaline phosphatase at a rate proportional to the activity of alkaline phosphatase to release the corresponding azo dye. Can be used as In particular, a compound in which R ⁴ and R ⁵ are hydrogen atoms in the general formula (I) is easily available as a raw material, and the visible absorption spectrum of a dye released as a result of an enzymatic reaction has a large absorption maximum wavelength and a large molecular absorption coefficient. It is preferable because it is large. Next, specific examples of the compounds represented by the above general formula (I) or (II) are shown, but the scope of the present invention is not limited to these compounds.

[Chemical 3]

[Chemical 4]

[Chemical 5]

[Chemical 6]

[Chemical 7] First, the aromatic amine A-NH ₂ (III) and the phenol derivative H-B-OH are diazo-coupled by a conventional method to synthesize an azo dye (A-N = N-B-OH) (IV). Since A has a strong electron-withdrawing group such as a methylsulfonyl group and a nitro group, the so-called nitrosyl sulfuric acid method (for example, SR Sandler and W. Karo [[ Organic Function
nal Group Preparations "No. 2
Volume, 295 pages (1971 Academic Pre
Specific examples are described in (issued by ss). ) Is preferably used. Then, the azo dye (IV) is reacted with a large excess of phosphorus oxychloride in the presence of pyridine to form a phosphorodichloridate (V), and then (V) is reacted with water to form a phosphoric acid monoester (I ′). And In the reaction for obtaining (V) from (IV), 3 times or more, preferably 3 to 10 times, mol of phosphorus oxychloride is added to acetone, trimethylphosphoric acid or a mixed solvent of acetone and trimethylphosphoric acid in (IV). At 2 to 50
A solution diluted to 1 ×, preferably 5 to 10 times, at −15 ° C. to 10 ° C., preferably −10 ° C. to 0 ° C., (I
V) is added dropwise to a solution of pyridine, pyridine-containing acetone, pyridine-containing trimethylphosphoric acid, or a mixed solvent of pyridine-containing acetone and trimethylphosphoric acid, to thereby produce almost no by-products (IV ) Can be converted to (V). It should be noted that a tertiary amine having a stronger basicity than pyridine such as triethylamine may be added to the solution of (IV) instead of pyridine. The formation of (V) can be confirmed by the appearance of spots having a smaller Rf value than that of (IV) in silica gel thin layer chromatography. Conventionally, aryl phosphorodichloridates such as p-nitrophenyl phosphorodichloridate have been synthesized by heating and refluxing phosphorus oxychloride and phenol in the presence of potassium chloride and the like. Specific examples thereof include, for example, Mukaiyama and Hashimoto, Bulletin of
heChemical Society of Japan
An magazine, Vol. 44, page 196 (published in 1971). However, under such violent conditions, the above compound (V) was not efficiently produced. When converting (V) to (I ′), water may be added to the reaction solution without isolating (V). However, since (I ') may decompose under acidic conditions, a tertiary amine such as pyridine capable of trapping hydrogen chloride generated during the reaction of (V) with water is added or it is insoluble in water such as ethyl acetate. A preferable result may be obtained by making a water-oil two-layer system coexist with another solvent. A solution of concentrated sodium hydroxide or potassium hydroxide was added to the thus obtained (I ') to dissolve it, the pH was adjusted to about 10, and the insoluble portion was filtered off to obtain a solution of about 10 to 20 times its volume. The crystal of (I) or (II) can be obtained by adding dropwise to ethanol with stirring. The compound (I) or (II) thus obtained can be purified by dissolving it in a small amount of water, injecting it into ethanol and crystallizing. The crystals obtained by this method often contain several molecules of crystal water per molecule. The compound of the present invention represented by the above general formula (I) or (II) synthesized as described above is excellent as a substrate for quantifying alkaline phosphatase. That is, the compound represented by the above general formula (I) or (II) can be produced by the action of (1) alkaline phosphatase.
It is hydrolyzed at a rate proportional to the activity of alkaline phosphatase to give azophenol dyes. (2) These dyes have been widely used in the prior art, and coloration in the method using disodium p-nitrophenylphosphate as a substrate. Since it has a maximum absorption of visible light on the longer wavelength side than p-nitrophenol, which is a dye, it is less susceptible to interference by colored substances such as blood dye, and (3) and these dyes are water-soluble, so the enzyme When used as a substrate for alkaline phosphatase quantification, it does not cause aggregation or precipitation during the activity measurement, and thus the degree of freedom in setting quantitative test conditions is high, and there are no difficulties in synthesis as shown in (4) Synthesis example. As mentioned at the beginning of the description, phthalein phosphate, thymolphthalein phosphate, or thymol blue monophosphate Isolation purification of the compounds in the synthesis of a complex, in contrast, can be purified by conventional crystallization. For quantifying alkaline phosphatase using the compound of the present invention, the Bessey-Lowry method (Journal of Biological Ch.
It is described in the magazine, 164, 321 pages, published in 1946. ), Using the compound of the present invention in place of p-nitrophenyl phosphate disodium,
Instead of determining the amount of p-nitrophenol resulting from the enzymatic reaction in the Bessey-Lowry method, the amount of azo dye A-N = N-B-OH generated from the compound of the present invention may be determined. For example, first, the compound represented by the general formula (I) or (II) is dissolved in a buffer solution having a pH of about 10 to prepare a substrate solution. At this time, it is preferable to add an enzyme activator (eg, magnesium chloride) to the substrate solution. Next, a liquid containing alkaline phosphatase, which is the substance to be measured, is added to the substrate liquid to carry out the reaction. At this time, the compound represented by the general formula (I) or (II) is preferably present in the substrate solution at a concentration of 1 to 20 mmol / liter. In addition, the general formula (I)
Alternatively, the compound represented by (II) is used in a large excess with respect to alkaline phosphatase, that is, in a molar ratio of 10
It is preferably present twice or more. The above reaction is 10
10 to 50 ° C, more preferably 30 to 40 ° C.
~ 60 minutes. Then, if necessary, the pH of the solution is increased or decreased by adding a base or an acid, and in some cases, an organic solvent such as DMF (dimethylformamide) or methylcellosolve is added to stop the reaction. At this time, the concentration of the azo dye thus formed is measured by a spectrophotometer or the like, and if necessary, alkaline phosphatase is quantified from the calibration curve. The colorimetric wavelength may be set near the absorption maximum wavelength of the dye, but it is also possible to shift the absorption maximum wavelength to the long wavelength side by adding cyclodextrin, preferably α-cyclodextrin, at the time of colorimetry. Is. At this time, the amount of cyclodextrins added was 1 ml of the colorimetric liquid.
The amount is 0.1 mg to 20 mg, preferably 1 mg to 10 mg. Next, among the compounds represented by the general formula (I) or (II) of the present invention, a synthesis example of the compound 3 will be shown. Compounds other than Compound 3 can be easily synthesized based on the above-mentioned synthetic method and in accordance with the following description. Synthesis example: Synthesis of compound 3 Phosphorus oxychloride 4 ml and acetone 20 ml were mixed-
Cooled to 8 ° C. To this solution, 2 g of 4- (2-methanesulfonyl-4-nitrophenylazo) phenol was added with 20 ml of acetone, 10 ml of trimethylphosphoric acid, and 8 parts of pyridine.
ml, and a solution of triethylamine in 0.5 ml was added dropwise over 25 minutes. During this time, the reaction solution was cooled from the outside so that the temperature did not exceed 0 ° C. 30 more
After continuing stirring at 0 ° C. or less for 30 minutes, 30 ml of water was added dropwise. After stirring at room temperature for 10 minutes, the mixture was poured into 200 ml of a saturated saline solution and ice, and the resulting crystals were collected by filtration.
20 ml of a 1N aqueous sodium hydroxide solution was added to the crystals to adjust the pH to about 10. After filtering off the insoluble portion, the filtrate was poured into 150 ml of ethanol, and the generated crystals were collected by filtration, washed with ethanol, and dried under reduced pressure. Yield 2 g, λ _max ^{0.02 N-NaOH} 382 nm Next, a specific example of a method for quantifying alkaline phosphatase using the compound of the present invention will be shown. Reference example 1. Preparation of substrate solution: Compound 3 crystal (35.05 mg) in 10 m
It was dissolved in 1 glycine buffer (pH 10, containing 0.5 mmole / liter of magnesium chloride). Preparation of Enzyme Solution: A solution prepared by dissolving about 6 mg of alkaline phosphatase Type I of Sigma Chemical Co. in 20 ml of distilled water and a solution prepared by diluting this solution 2 times and 4 times 3 times.
Kind prepared. Procedure: 1 ml of the substrate solution was placed in a test tube and heated to 37 ° C for 3 minutes. The enzyme solution (0.1 ml) was added, and the mixture was heated at 37 ° C. for 30 minutes for reaction, and then the reaction was stopped by adding 0.02 N sodium hydroxide aqueous solution (10 ml). The absorbance (ΔOD) was measured at 520 nm using a blind solution treated in exactly the same manner except that distilled water was used instead of the enzyme solution. In this way, measurement was performed for each of the three types of enzyme solutions having different concentrations. Be for the same enzyme solutions as these
When the ssey-Lowry method was applied, the absorbance (ΔOD) at 410 nm using the blind solution as a control was measured. The operation of the Bessey-Lowry method was carried out according to the above compound 3
The procedure is exactly the same as that described above, except that 20 mg of sodium p-nitrophenylphosphate is used instead of 35.05 mg of the crystal of Example 1 above to perform colorimetry at 410 nm. The relationship between the ΔOD values obtained by these two methods is shown in FIG. The following is clear from FIG. (1) In proportion to the enzyme concentration, that is, Bessey-
Since ΔOD changes in proportion to the value obtained by the Lowry method, the activity of the enzyme can be determined from the value of ΔOD. (2) ΔOD when using Compound 3 as a substrate is Bess
Since the change is at a rate larger than the ΔOD of the ey-Lowry method (the slope of the straight line is 1 or more), the compound 3 as a substrate has higher sensitivity than the Bessey-Lowry method. (3) When Compound 3 is used as a substrate, colorimetry can be performed at 520 nm, and therefore Bessey-L that is colorimetric at 410 nm
Less likely to be disturbed by colored substances such as pigments in blood than the owry method. As described above, it is understood that the compound of the present invention is a compound suitable as a substrate for quantifying alkaline phosphatase. Reference example 2. The procedure of Reference Example 1 was repeated, except that 48.18 mg of the crystal of Compound 12 was used instead of 35.05 mg of the crystal of Compound 3 of Reference Example 1, and the absorbance was measured at 644 nm instead of 520 nm. The results are shown in Fig. 2. The following is clear from FIG. (1) In proportion to the enzyme concentration, that is, Bessey-L
Since ΔOD changes in proportion to the value by the owry method, Δ
The activity of the enzyme can be determined from the value of OD. (2) When compound 12 is used as a substrate, 644 nm
Color comparison can be done at 410nm.
-It is less likely to be disturbed by colored substances such as pigments in blood than the Lowry method. As described above, it is understood that the compound of the present invention is a compound suitable as a substrate for quantifying alkaline phosphatase.

[Brief description of drawings]

FIG. 1 is a graph showing the results when alkaline phosphatase was measured using Compound 3.

FIG. 2 is a graph showing the results when alkaline phosphatase was measured using Compound 12. 1 and 2, the horizontal axis is the absorbance (ΔOD) when the Bessey-Lowry method is used, and the vertical axis is the absorbance (ΔOD) when the compound of the present invention is used as a substrate.

Claims (1)

[Claims] 1. A phosphoric acid ester represented by the general formula (I) or (II). [Chemical 1] (In the formula, M is a hydrogen atom, a sodium atom or a potassium atom, one of R ¹ and R ² is a methylsulfonyl group,
The other of R ¹ and R ² is a nitro group, R ³ is a chlorine atom or a hydrogen atom, R ⁴ is a hydrogen atom, a hydroxyl group, a methyl group or a methylcarbonylamino group, and R ⁵ is a hydrogen atom or a chlorine atom. And R ⁶ is a methyl group or 3
A sulfamoylphenyl group. )