JPS603396B2 - Tetrazolium salt compound and spectrophotometric determination of dehydrogenase using the compound - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel tetrazolium salt compound and a method for spectrophotometric determination of dehydrogenase using the compound. Concerning spectrophotometric determination.

However, the meanings of the symbols in the formula are as follows. Y: A straight chain type having 2 to 4 carbon atoms, or an alkylene group that may have a side chain and, if necessary, one hydroxyl group.

RI: an alkyl group having 1 to 4 carbon atoms or a hydroxyethyl group.

R2: an alkyl group having 1 to 4 carbon atoms and optionally having 1 to 2 hydroxyl groups or 1 phenyl group.

×: Chlorine atom or bromine atom.

W: hydrogen atom.

Z: 4,5-dimethyl-2-thiazolyl group.

In addition, in the benzene ring of The substitution position is the 3rd or 4th position.

The compound having the above-mentioned single-pronged formula (1) according to the present invention is a novel substance that has not been previously reported, and has the great feature that it can be used for the determination of dehydrogenases because it accepts hydrogen and produces formazan.

That is, a conventional tetrazo IJum salt compound has been used for quantifying dehydrogenase, but the formazan produced by reduction thereof is not water-soluble and is therefore quite inconvenient in practice. By the way, dehydrogenase quantification using formazan production reaction is now widely used, especially in clinical testing, so in reality, quantitative operations are performed using automatic analyzers. Opportunities are increasing.

However, when known tetrazolium salts are used, the formazan generated during the operation is insoluble in water, and this can adhere to the inner wall of the analyzer tube or become clogged, which poses a major hindrance to the automation of testing. It has become. In order to overcome these drawbacks, the first step is to create water-soluble formazan. The present invention successfully solves this problem. Below, we will explain in a little more detail how water-soluble formazan, which is produced by accepting hydrogen like the compound of the present invention, is useful in clinical tests. Dehydrogenases, such as lactate dehydrogenase (hereinafter abbreviated as "LDH").

), alcohol dehydrogenase, glutamic acid dehydrogenase, etc., have been quantitatively analyzed using tetrazolium salt compounds. That is, the tetrazolium salt compound generates formazan by accepting hydrogen liberated by the action of these various dehydrogenases via an intermediate electron carrier. Dehydrogenase can be quantified by measuring the absorbance of the formazan produced here. By the way, among these dehydrogenases, LDH is distributed in all body cells, and is particularly present in large quantities in cardiac muscle, liver, skeletal muscle, kidney, etc., and is associated with myocardial infarction, malignant seed injury, liver disease, progressive muscle atrophy, and blood vessels. It is known that serum LDH activity increases markedly in cases of diseases such as internal hemolysis and megaloblastic anemia. Therefore, by measuring LDH activity in the blood, clinically, extremely meaningful knowledge for the diagnosis of the above-mentioned diseases can be obtained. As mentioned above, the secondary compound is a tetrazolium salt compound, such as 3,3'-(3,3-dimethoxy4,4'
-Bipheni, rylene)-bis[2-(4-nitrophenyl)-5-phenyl-pantetrazoIJum salt compound] (
Below is “Nito.

It is abbreviated as "TB". ) are commonly used as hydrogen acceptors for this purpose. However, the formazan produced when this nitro-TB accepts hydrogen is completely insoluble in water, which poses various practical problems. In particular, with the spread of automatic analysis methods, autoanalyzers have been frequently used, but in this case, the formazan produced deposits on the analyzer tube, etc., and has a fatal drawback in that it interferes with subsequent operations. Therefore, currently, the insoluble formazan is dispersed using a dispersant such as a surfactant and then the absorbance is determined, but this not only complicates the operation but also causes errors in the measured values. This was an extremely undesirable cause. In order to eliminate these drawbacks, it is necessary to use a compound in which the produced formazan is easily soluble in water.

For that purpose, compounds such as 3-(4-iodophenyl)-2-(4-nitrophenyl)-15-phenyl-tetrazolium chloride, which slightly increase the water solubility of the formed formazan, may also be used. However, for the purpose of spectrophotometric measurement, even this formazan has too little water solubility to be practically satisfactory. As a result of various studies conducted by the present inventors on compounds that produce formazan with good solubility and stable measurement values, the present inventors have found that the compound having the general formula (1) is a hydrogen acceptor with excellent water solubility. They discovered this and completed the present invention.

In the past, various methods for making compounds water-soluble have been studied, and among them, attempts have been made to some extent to improve water solubility by introducing sulfonic acid groups.

However, such sulfonic acid converters have problems such as being difficult to purify and, in the form of tetrazolium salts, having rather low water solubility. On the other hand, the compound of the present invention having a quaternary ammonium salt in its side chain has the extremely advantageous feature that the formazan produced from the compound is highly water-soluble. A quantitative method that is worry-free and provides stable measurement values has been established. The compound having the general formula (1) according to the present invention can be produced by a conventional method. For example, 3 (or 4)-hydroxybenzaldehyde, epichlorohydrin under basic conditions,
Alternatively, a corresponding 3 (or 4)-alkoxybenzaldehyde is first synthesized by reacting an alkyl dihalide having 2 to 4 carbon atoms. In the case of an alkoxybenzaldehyde compound having oxirane pseudo, it may be reacted with hydrochloric acid or bromic acid under water to form a halohydrin compound. The alkoxybenzaldehydes obtained here are then treated with phenylhydrazines to form the corresponding hydrazone compounds. Among these, the hydrazone compound having an oxirane ring is reacted with a dialkylamine to form a hydrazone compound having a tertiary amine in the side chain. Further, a hydrazone compound having a halogen in the side chain may be reacted with a dialkylamine. Next, the hydrazone compound was subjected to a coupling reaction with a diazonium salt of 4,5-dimethylthiazole to form formazan under basic conditions, and the formazan having a tertiary amine in the side chain was combined with an alkyl halide and a halogen in the side chain. Formazan is reacted with tertiary amine to synthesize the corresponding quaternary ammonium salt compound.

Next, such formazan having a quaternary ammonium salt in its side chain is oxidized using an oxidizing agent such as n-butyl nitrite or N-bromosuccinimide (hereinafter abbreviated as "N-mo") to obtain the desired product. A tetrazolium salt compound is obtained. These details will be described more specifically in the examples. Next, a method for spectrophotometric determination of dehydrogenase using the thus obtained compound having the general formula (1) will be explained.

That is, a dehydrogenase is brought into contact with a substrate corresponding to the enzyme in a phosphate buffer, Tris-HCl buffer, or the like. The hydrogen generated here is once accepted by nicotinamide adenine dinucleotide (hereinafter abbreviated as "NAD"),
Next, diafluorase and phenazine methosulfate (
Hereinafter, it will be abbreviated as "PMS". ) Hydrogen is accepted by the tetrazolium salt compound of the present invention through an intermediate electron carrier such as ), thereby producing formazan with a high degree of water solubility not found in conventional formazan. Therefore, the activity value of the target enzyme can be accurately quantified only by directly measuring the absorbance of the resulting reaction solution. The present invention will be described in more detail with reference to Examples below, but the present invention is not limited to the following Examples unless it exceeds the gist thereof.

Example 1 (In general formula (1), Y is ethylene, R1 and R2 are ethyl groups, × is bromine atom, W is hydrogen atom, Z is 4,5-dimethyl-2-thiazolyl group, and the substituted alkoxy group is a benzene ring Synthesis method of a compound substituted at the 4-position.

) 35 mm of 4-hydroxybenzaldehyde and 270 mm of 1,2-dipromoethane were dissolved in tetrahydrofuran,
A sodium hydroxide solution is added to the mixture, heated to reflux, and then distilled under reduced pressure to obtain 41.4 ml of 4-(2-promoethoxy)pensaldehyde.

Next, 40 parts of this 4-(2-promoethoxy)penzaldehyde and 18.9 parts of phenylhydrazine are mixed while heating in methanol to synthesize 42.6 parts of 4-(2-promoethoxy)penzaldehyde phenylhydrazone. The obtained hydrazone 18 was dissolved in a mixed solvent of tetrahydrofuran and methanol, and a potassium hydroxide solution was added while cooling. An aqueous solution of diazonium hydrochloride synthesized using 2-amino-4,5-dimethylthiazole hydrochloride, hydrochloric acid, and sodium nitrite was added little by little to the mixture and allowed to stand for 2 hours. Methanol was added to the reaction solution, and the mixture was allowed to stand overnight and filtered to obtain 3-(4,5-dimethyl-2-thiazolyl)-12-phenyl-5-[4-(2-promoethoxy)phenyl]formazane 21. The resulting formazan 20 ml was dissolved in a mixed solvent of triethylamine and tetrahydrofuran, heated under reflux, and the newly formed crystals were collected in a furnace to obtain 13.4 ml of the quaternary ammonium salt of formazan side chain. Dissolve 12 parts of this quaternary ammonium salt in methanol, add 4.6 parts of N speck little by little, and mash. Activated carbon is added to the reaction solution, filtered through a furnace, and the furnace liquid is concentrated under reduced pressure to obtain the desired tetrazolium bromide. Melting point 167-1
71℃ (decomposition) Elemental analysis value (%) C several days 34N60SBr20 days
N theoretical value 48.91 5.37 13.16 actual value 49.38 5.41 12.961 days -
NMR (mu DMSO) 6 chest (TMS) 1.10 (t,
J=6.7HZ, group) 3.12-4.35 (m, 1 book) 7.08-7.75 (m, cape) 8.06-8,5
1 (m, melon) IR (skin-1) 2870 (1 CH) 1585 (-C=N-) 1460
(1CH) 1220(1COC-) Example 2 (In the general formula (1), Y is butylene, RI is a methyl group, R2 is a penzyl group, X is a bromine atom, W is a hydrogen atom, Z
is a 4,5-dimethyl-2-thiazolyl group and a substituted alkoxy group is substituted at the 3-position of the benzene ring.

) 3-Hydroxybenzaldehyde (46 mm) and 1,4-dichlorobutane (240 mm) were dissolved in tetrahydrofuran, a sodium hydroxide solution was added thereto, the mixture was heated to reflux, and then distilled under reduced pressure to obtain 3-(4-chlorobutoxy)penzaldehyde (45 mm). .

Next, 10 days of this 3-(4-chlorobutoxy)penzaldehyde and 5.1 days of phenylhydrazine were heated and mixed in methanol, allowed to stand overnight, and then cooled under ice. was dissolved in methanol and reacted, and after gradually returning to room temperature, the solvent was distilled off, and 3-
(4-dimethylaminoptoxy)penzaldehyde phenylhydrazone 13.5 hours is obtained. The obtained hydrazone 6 is dissolved in methanol, and potassium hydroxide solution is added while cooling. An aqueous solution of diazonium hydrochloride synthesized using 2-amino-4,5-dimethylthiazole hydrochloride, hydrochloric acid, and nitrous acid was gradually added dropwise to the solution for 2 hours. Add water to the reaction solution and leave it overnight, then filter it in an oven.
4.6 hours of 3-(4,5-dimethyl-2-thiazol)-12-phenyl-5-[3-(4-dimethylaminobutoxy)phenyl]formazane are obtained. Penzyl bromide 25
Dissolve the formazan and formazan in tetrahydrofuran, add oxidized steel powder, heat to reflux, collect the crystals in a furnace, dissolve in methanol and filter, and concentrate the furnace liquid under reduced pressure to obtain the quaternary ammonium salt of the formazan side chain.2. Get 5 evenings. This quaternary ammonium salt was dissolved in methanol, and 0.6 N spots were added to it.
9 nights later, I was in tears. Activated carbon is added to the reaction solution, filtered through a furnace, and the furnace liquid is concentrated to obtain the desired tetrazolium bromide. Melting point 159-163oo (decomposition) Elemental analysis value (%) CM date 26N60SBr2, ship O○
H N theoretical value 51.82 5.33 11.
70 Actual value 51.77 5.21 11.94I
H-NMR (66-DMSO) Hexapod (TMS) 1.0
5-1.72 (m, mountain H) 31.5-4.46 (m,
19H) 4.91 (S, 2H) 6.85-7.46
(m, mountain H) 7.52-8.00 (m, group) 8.05
~8.51 (m, mark) IR (skin-1) 2 balls 0 (1 CH)
1600 (-C NiN-1) 1465 (1 CH) 121
5 (One COC-) Example 3 (Spectrophotometric determination of dehydrogenase using the compound obtained in Example 2).

) 370 pre-flooded glycine-lactate solution (glycine = 0.72%, dl-lactic acid = 0.42%, pH = 9.
6) Add standard serum pre-flooded with the same heating for 30 minutes to 0 to 82. Hada U/'s addition and side worship.

After 1 minute, 0.0706% aqueous solution of the compound obtained in Example 2 was added and stirred, and after another 1 minute, NAD-PMS was added.
Solution (NAD = 2%, PMS = 0.2%, morning serum albumin 5 = 0.15%, pH = 7.4 phosphate buffer) Add 0.1 ml and mix to 370. Hold for exactly 8 minutes.

Immediately add 1 part of 0.1N hydrochloric acid, and measure the absorbance using a 51-point tube. Purified water was used instead of serum as a control. As a result, as shown in FIG. 1, a calibration curve with good linearity passing through the origin was obtained, and thereby the enzyme activity of serum can be determined accurately. Example 4 (In general formula (1), Y is 2-hydroxypropylene, R1, R2 are 2-hydroxyethyl group, × is bromine atom, W is hydrogen atom, Z is 4,5-dimethyl-2-thiazolyl group, A method for synthesizing a compound in which a substituted alkoxy group is substituted at the 4-position of a benzene ring.

) 4-hydroxybenzaldehyde (114 mm) and epichlorohydrin (170 mm) were dissolved in tetrahydrofuran, a sodium hydroxide solution was added thereto, the mixture was heated to reflux, and then distilled under reduced pressure to obtain 4-oxiranemethoxybenzaldehyde (1).
Get 00 evenings. Next, 25 hours of this 4-oxirane methoxybenzaldehyde and 15.2 hours of phenylhydrazine are heated and mixed in methanol to synthesize 4-oxirane methoxybenzaldehyde phenylhydrazone 312.
15 parts of this hydrazone was dispersed in methanol, 18 parts of diethanolamine was added while stirring, and after heating, methanol was further added, and a potassium hydroxide solution was added while cooling. An aqueous solution of diazonium hydrochloride synthesized using 9.5 g of 2-amino-4,5-dimethylthiazole hydrochloride, hydrochloric acid, and sodium nitrite was added dropwise little by little to the mixture. Water was added to the reaction solution, left overnight and filtered.
5-dimethyl-2-thiazolyl)-2-phenyl-5-{4-[2-hydroxy-3-di(2-hydroxyethyl)aminopropoxy]phenyl}formazan 18.3
Get the evening. The obtained formazan 11 is dissolved in a mixed solvent of ethylene bromohydrin and tetrahydrofuran, concentrated by heating, and tetrahydrofuran is added thereto to obtain a quaternary ammonium salt of formazan side chain 9.6. Dissolve 8.5 liters of this quaternary ammonium salt in methanol,
Add NBS 3.1 evening to it little by little and pray. Activated carbon is added to the reaction solution, heated in a furnace, and the solution is concentrated to obtain the desired tetrazolium bromide. Melting point 116-1220 Elemental analysis value (%) C Kuhi 36N505Br20 days
N Theoretical value 48.37 5.41 10.45 Actual value 48.74 5.56 10.021 days -
NMR (mu DMSO) hexapod (TMS) 3,26-4.
63 (m, 2 ho) 7.02~7,59 (m, 'H)
8.00~8.45 (m, porridge) IR (Ki-1> 3240 (1 OH) 2875 (1 CH) 1600 (
1C=N-)1450<-CH)

[Brief explanation of the drawing]

FIG. 1 shows 51.51 according to Example 3. The relationship between absorbance and LDH activity value at trace m is shown. Figure 1

Claims (1)

[Claims] 1. A tetrazolium salt compound represented by the general formula ▲ Numerical formula, chemical formula, table, etc. ▼. However, the meanings of the symbols in the formula are as follows. Y: A straight chain type having 2 to 4 carbon atoms, or an alkylene group that may have a side chain and, if necessary, one hydroxyl group. R^1: an alkyl group having 1 to 4 carbon atoms or a hydroxyethyl group. R^2: An alkyl group having 1 to 4 carbon atoms and optionally having 1 to 2 hydroxyl groups or 1 phenyl group. X: chlorine atom or bromine atom. W: hydrogen atom. Z: 4,5-dimethyl-2-thiazolyl group. In addition, ▲There are mathematical formulas, chemical formulas, tables, etc.▼ The substitution position on the benzene ring is the 3rd or 4th position. 2 Spectrophotometric determination of dehydrogenase using a tetrazolium salt compound represented by the general formula ▲Mathematical formula, chemical formula, table, etc.▼. However, the meanings of the symbols in the formula are as follows. Y: A straight chain type having 2 to 4 carbon atoms, or an alkylene group that may have a side chain and, if necessary, one hydroxyl group. R^1: an alkyl group having 1 to 4 carbon atoms or a hydroxyethyl group. R^2: An alkyl group having 1 to 4 carbon atoms and optionally having 1 to 2 hydroxyl groups or 1 phenyl group. X: chlorine atom or bromine atom. W: hydrogen atom. Z: 4,5-dimethyl-2-thiazolyl group. In addition, ▲There are mathematical formulas, chemical formulas, tables, etc.▼ The substitution position on the benzene ring is the 3rd or 4th position.