JPS58113181A - Tetrazolium salt compound and absorption photometry of dehydrogenase using said compound - Google Patents

Abstract

NEW MATERIAL:The compound of formulaI[Y is 2-4C alkylene which may have side chain and if necessary one OH group; R<1> is 1-4C alkyl or hydroxyethyl; R<2> is 1-4C alkyl which may have 1-2 OH groups or one phenyl group; X is Cl or Br; W is H (when Z is 4,5-dimethyl-2-thiazolyl) or NO2 (when Z is 4- iodophenyl); the group of formula II is at the 3-4 position of the benzene ring]. EXAMPLE:3-( 4-Iodophenyl )-2-( 4-nitrophenyl )-5-[4-( 2-hydroxy-3-diethylamino- propoxy)phenyl]-2H-tetrazolium salt compound. USE:A hydrogen acceptor for absorption photometry of a dehydrogenase, giving formazan having high water-solubility and giving stable measured value. PROCESS:The compound of formulaIis prepared by reacting a formazan having tertiary amine group as a side chain with an alkyl halide, and oxidizing the resultant quaternary ammonium salt.

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 a method for spectrophotometric determination of dehydrogenase. However, the meanings of the symbols in the formula are as follows.

=3- Y: A straight chain type having 2 to 4 carbon atoms, or having a side chain.

An alkylene group which may further have one hydroxyl group if necessary.

RI: Alkyl group having 1 to 4 carbon atoms, still hydroxyethyl group.

R1: Alky/''M 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 or nitro group.

Z: 4.5-dimethy) when W is a hydrogen atom; 4-iodophenyl group when W is a canitro group;

Or 4th place.

The compound having the above-mentioned general formula (1+) according to the present invention is a novel compound that has not been described in any literature, and has the great feature that it can be used for dehydrogenase electric resistance stitching because it accepts hydrogen and produces formazan. That is, a conventional tetrazolium salt compound has been used for the quantitative determination of hydrogen enzyme, but the formazan produced by distilling this compound is practically inconvenient due to its non-water bath property.

4- By the way, dehydrogenase quantification using the formazan production reaction is widely used today, especially in the field of clinical testing. Opportunities to perform operations are dwindling. However, when known tetrazolium salts are used, the formazan generated during the operation is insoluble in water, and this adheres to or deposits on the inner wall of the analyzer tube, which poses a major hindrance to the automation of testing. ing. 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 how useful the fact that formazan, which is produced by accepting hydrogen like the 9 compounds of the present invention, is water-bathable in clinical tests. explain in detail.

Dehydrogenases, such as lactate dehydrogenase (hereinafter "LDH")
Abbreviated as (alco-)V dehydrogenase, glutamic acid dehydrogenase, etc., have been analyzed using tetrazolium salt compounds. In other words, tetrazolium salt compounds generate formazan by accepting hydrogen liberated by the action of these various dehydrogenases via intermediate i (child carrier).Then, the absorbance of the formazan thus generated is measured. By the way, dehydrogenase can be quantified by
DH is distributed in all body axes 11ii1, especially the U muscle.

Predominantly present in the liver, skeletal muscles, kidneys, etc., and U muscle infarction,
It is known that serum LDH activity increases markedly in cases of diseases such as malignant tumors, liver diseases, progressive muscular atrophy, intravascular hyperemia, and megaloblastic anemia. Therefore, clinically, by measuring LDH activity in the blood, extremely significant knowledge for the diagnosis of the above-mentioned diseases can be obtained.

Conventionally, as mentioned above, tetrazolium salt compounds, such as 3.3'-(3,3'-dimethoxy-4,4'-biphenylylene)-bis[2-(4-nitrophenyl/V)
-5-phenyl-2H tetrazolium salt compound] (hereinafter abbreviated as nitro-TBJ) is commonly used as a futile hydrogen acceptor for this purpose.7 However, this nitro-TB does not accept hydrogen. The formazan produced in this process is completely insoluble in water, which poses various practical problems.

In particular, with the spread of automatic analysis methods, autoanalyzers are frequently used, but in this case, the formazan produced sinks into the analyzer's cheapness, causing problems in subsequent operations, which is a fatal drawback. . Therefore, at present, the insoluble formazan is dispersed using a dispersant such as a surfactant and then the absorbance is determined.

Is this just an operation? In order to eliminate these drawbacks, which are extremely undesirable as they not only result in 11 conversion, but also cause errors in side measurements, it is necessary to use a compound in which the formed formazan is easily soluble in water. be. For that purpose.

7'c,! :, tHa3- (4-yo F phenyl)-
2-(4-nitrophenyl)-5-pheny/L'-2H
Tetrazolium? Compounds such as X(IJI) that slightly increase the water solubility of the formed formazan are also used,
For the purpose of spectrophotometer measurement, the water solubility of the formazan is still too low to be practically satisfactory.

The present inventors have conducted various studies on compounds that produce formazan, which has good bath dissolubility and provides a stable initial value.

The present invention was completed by discovering that the compound having the general formula (1) is a water-soluble, suspended hydrogen acceptor.

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 derivatives have problems such as being difficult to purify and having rather low water solubility in the form of tetrazolium salts. This includes 7i1. ．． The compound of the present invention has a quaternary ammonium salt in its side chain.

7- Since the formazan that will be produced has the extremely advantageous feature of being highly water-soluble, it is possible to develop a quantitative method that allows stable measurement values to be obtained without worrying about adhesion to automatic analyzers or precipitation by using this compound for one month. It has been established.

The compound having the general formula (1) according to the present invention can be prepared by a conventional method. For example, 3 (or 4)-hydroxybenzaldehyde with 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 containing an oxirane ring, it may be reacted with hydrochloric acid or bromic acid under water cooling to form a halohydrin compound. The alkoxybenzaldehydes obtained here are then reacted with phenylhydrazines to form the corresponding hydrazone compounds. Among these, hydrazone compounds have oxirane F stars.

It 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 treated with 4-iodobenzenediazonium salt or 4,5-
After coupling reaction with 8-diazonium salt of dimethylthiazole to form formazan, formazan having a tertiary amine in the side chain is reacted with an alkyl halide, and formazan having a halogen in the side chain is reacted with a tertiary amine. A 4R ammonium salt compound is synthesized.

Next, such formazan having a quaternary ammonium salt in its side chain is oxidized using an oxidizing agent such as n-butyl nitrate or N-bromosuccinimide (hereinafter abbreviated as "NBsj") to obtain the desired product. A tetrazolium compound is obtained.The details of these compounds will be described in more detail in the examples.

Next, the compound having the general formula (1) thus obtained is brought into contact with a substrate. The hydrogen produced here is
Nicotinamide adenine nucleotide (hereinafter JN, A
Abbreviated as DJ. ), and hydrogen is then accepted by the tetrazolium salt compound of 9 Honkomei through an intermediate carrier such as cyafluorase or phenazine methosulfate (hereinafter abbreviated as [PMSj). Here, conventional formazan has Produces formazan with an unacceptably high degree of water solubility. Therefore, i of the resulting reaction solution
Only by directly measuring the W likelihood, the activity value of the target enzyme can be accurately quantified.

The present invention will be explained in more detail with reference to Examples below.

The present invention is not limited to the following embodiments without exceeding the gist thereof.

Example 1 (In the general formula (1), Y is 2-hydroxypropylene R'' is an ethyl group, R is a 2-hydroxyethyl group, X is a chlorine atom, W is a nitro group, and 2 is a 4-iodophenyl group) , a method for synthesizing a compound in which a substituted alkoxy group is substituted at the 4-position of the benzene ring.) 114 g of 4-hydroxybenzaldehyde and 100 f of ebichlorohydrosilane methoxybenzaldehyde are obtained. Next, 50 f of this 4-oxirane methoxybenzaldehyde and 4-nitro Phenylhydrazine 43f was heated and mixed in methanol.

Synthesize 4-oxirane methoxybenzaldehyde-4-nitrophenylhydrazone 85.9. 4 oy of this hydrazone is dispersed in methanol, 3 o 9 of diethylamine is added with stirring, heated and concentrated under reduced pressure to obtain 4'l of 4-(2-hydroquine-3-diethylaminopropoxy)benzaldehyde-4-nitrophenylhydrazone. The obtained hydrazone 18f was dissolved in tetrahydrofuran, a potassium hydroxide solution was added thereto, and while cooling, an aqueous solution of 4-iodobenzenediazonium hydrochloride synthesized using 4-iodoaniline 105f, hydrochloric acid, and sodium nitrite was gradually added dropwise. and stir for 2 hours. Water was added to the reaction solution and filtered to give 3-(4-iodopheneAz)-2(4
-Nitrophenyl/l/)-5-(4-(2-hydroxy-3-diethylaminopropoxy)phenyl)formazan 155ff was obtained. The obtained formazan 12p was dissolved in a mixed solvent of ethylene chlorohydrin and tetrahydrofuran and heated in a refrigerator. Add methanol/I/ to the reaction m solution and f
FI 1t-5 and the furnace liquid was condensed to σ to obtain 8.59 quaternary ammonium salts of formazan side chains. 2. Dissolve 8 g of this quaternary ammonium salt in methanol, add hydrochloric acid to it, and stir at room temperature. Add 3.5p of 1lTi butynitrate zLz from the mixture. The reaction mixture is filtered and evaporated under reduced pressure to obtain the desired tetrazolium salt compound 7.5f.

Melting point 105-112°C (decomposition) Elemental analysis 11jL w) CAM B12 No Oa
C4ICHN Theoretical value 45,86 4.40 11.46 Actual value 46.18 4.35
11.021.25ft, J=6.7Hz, 6H13
, 15-4.68 (m, 13H) 7.00-7.63 (
m, 4H38, 15-8.601rn, 8H) IR (7
7f'1 3300(-CH) 2860(-CH) 16
00(-NOt11450(-CH) 1245(-
COC-) 850 (-Q-1 Example 2 (Spectrophotometric determination of dehydrogenase using the compound obtained in Example 1).

) Stir. 0.0 of the compound obtained in Example 1 after 1 minute.
Add 1 ml of 710% aqueous solution and stir, and after 1 minute add NAD-PMS solution 1 (NAD=2%, PMS-0.2%).
, bovine serum 7/L'bumin fraction 5=o15'1. PH=7
．． Add 0.1 ml of phosphate buffer (No. 4) and stir.
Hold at 37°C for exactly 8 minutes. Immediately o1 normal hydrochloric acid
ml and measure the absorbance at 495r+m. As a control, ice cubes were used instead of serum. 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 3 (in general formula (1), Y is ethylene, R'',
R' is an ethyl group, X is a bromine atom, W is a hydrogen atom, 2 is 4
．． A method for synthesizing a compound in which 5-dimethyp=2-thiazolyl group is substituted with a substituted alkoxy group at the 4-position of the benzene ring. ) 4-Hydroxybenzaldehyde 35FI and 270 g of 1,2-dibromoethane were dissolved in tetrahydrofuran,
A sodium hydroxide solution was added to the mixture, heated to reflux, and then distilled under reduced pressure to obtain 4=(2-bromoethoxy)benzaldehyde, il, 4ge.

Next, this 4-(2-bromoethoxy)benzaldehyde and 4Q fJa phenylhydrazine 18. 1 was heated and mixed in methanol to synthesize 426 g of 4-(2-bromoethoxy)benzaldehyde phenylhydrazone. The obtained hydrazone 18f was dissolved in a mixed solvent of tetrahydrofuran and methanol.

While cooling, add potassium hydroxide solution. 2 to this
-amino-4,5-dimethyp-thiazole hydrochloride 9.49
A diazonium hydrochloride aqueous solution synthesized using , hydrochloric acid, and sodium nitrite was added dropwise little by little, and the mixture was stirred for 2 hours. Add methanol to the reaction solution, leave it overnight for 11 t, filter, and add 3-
(4,5-dimethy/L'-2-thiazolyl)-2-phenyl-5-(4-(2-bromoethoxy)phenyl)formazan 211 is obtained.The obtained formazan 13-2Of is mixed with triethylamine and tetrahydrofuran. The crystals precipitated by heating and refluxing in a mixed solvent are counted.
13.4 fI of the quaternary ammonium salt of the formazan side chain is obtained. This quaternary ammonium salt 12f is dissolved in methanol, and NB54.61 is added little by little and stirred. Activated carbon is added to the reaction solution and filtered, and the p solution is compressed under reduced pressure to obtain the desired tetrazolium bromide.

Melting point 167-171℃ (decomposition) Elemental analysis value C? l;) C**&Na08Br2HN Theoretical value 48.91 5.37 13.16 Actual value
49.38 5.41 12.96'HNMR(da
-DM80) JP (TMS 11.10 (t
, J-6,7Hz, 9H) 3.12-4.
35 (m, 16H) 7.08-7.75 (m, 4H
) 8.06-8.51 (m, 5H) IR (4') 287 (H-CH) 1585 (-C=N-11460 (
-CH) 1220 (-COC-) 880 (-Q-1 Example 4 (Y in general formula (1) is 2-hydroxymethylethylene, R', R is a propyl group, X is a chlorine atom, W is a nitro group .

Z75E4-Iodophenyl group, a method for synthesizing a compound in which a substituted alkoxy group is substituted at the 3-position of the benzene ring,) 3-Hydroxybenzaldehyde 869 and epichlorohydrin 13011' are bathed in tetrahydrofuran, and sodium hydroxide'#4 solution is added to this. was added, heated to reflux, and then distilled under reduced pressure to obtain 3-oxiranemethquine benzaldehyde 75
get f. Next, 10 y of 3-oxirane methoxybenzaldehyde was slowly added to 14 m/of 64 acid without cooling.
After stirring M'Ft, chloroform was added, and the mixture was washed with cold diluted sodium hydrogen oxide solution and then with cold water.

The solvent is distilled off to obtain 10.5 p of 3-(2-chloro-3-hydroxypropoxy)benzaldehyde. This 3
-(2-chloro-3-hydroxyprophoki V)benzaldehyde 9y.

4-nitrophenylhydrazine 6.4F is heated and mixed in methanol to synthesize a-(2-chloro-3-hydroxypropoxy)benzaldehyde-4-nitrophenylhydrazone z, 411. This hydrazone 10.
4f is fermented in a mixed medium of tetrahydrofuran and methanol, a potassium hydroxide solution is added thereto, and the mixture is cooled. An aqueous solution of 4-iodobenzenediazonium hydrochloride synthesized using 4-iodoaniline 65f, hydrochloric acid and sodium nitrite was gradually added to the mixture and stirred. Water was added to the reaction solution, left overnight, and filtered to obtain 3-(4-iodophene).
V)-2-(4-nitropheni/1z)-5-(3-(
2-chloro-3-hydroxypropoxy)pheni/L/
Take 85 g of formazan. Obtained formazan 7.5
Dissolve g in a mixed solvent of tripropylamine and half-tetrahydrofuran and heat to reflux. Tetrahydrofuran is added to the reaction bath liquid and filtered to obtain 45.2p of quaternary ammonium in the formazan side chain. This quaternary ammonium salt 4y was dissolved in methanol, hydrochloric acid was added thereto, and the mixture was stirred at room temperature.

Then, add butylene nitrite/l/2.8f to this and react. Activated carbon is added to the reaction solution, filtered, and concentrated under reduced pressure to obtain 3.8f of the desired tetrazolium chloride.

Melting point 113-118°C (decomposition) Elemental analysis value (96) os+nsaNaoncgtr
, a.

CHN Theoretical value 48,01 5.33 10.84 Measured value 48,16 5.45 11.211H-NM
R(d, -DMSO1δ, (TMSll, 16(t, J
=6.6Hz, 9H) 2.15”2.25(m, 6H
1 turn 3.06~4.45+m, 18H16, 98~7.
60 (rn, 4H) 8.10-8.55 (m, 8H) 15- IR (cIR-'+ 3250 (-OH) 2880 (-CH) 1
605(-NOI11240(-COC-1860(-
Q-) Example 5 (Spectrophotometric determination method of dehydrogenase using the compound obtained in Example 4.) Good linearity was obtained through the origin as shown in Figure 2 by the same measurement procedure as in Example 2. A calibration curve was obtained. The compound was used as a 0.070% f4 solution, and the measurement was performed at 495 nm.

Example 6 (General formula (Y in 11 is butylene, R1 is methyl group.

R1 is a benzene/L/ group, X is a bromine atom, W is a hydrogen atom,
A method for synthesizing a compound in which 2 is a 4,5-dimethy)V-2-thiazolyl group and a substituted alkoxy group is substituted at the 3-position of the benzene ring. ) 3-hydroxybenzaldehyde 46f; 1.
4-Dichloro-(4-chlorobutoxy)benzaldehyde 459 is obtained.

Next, 10 y of this 3-(4-chlorobutoxy)benzaldehyde and 5.19 y of phenylhydrazine were humidified and mixed in methanol, left overnight, cooled with water, and while stirring, 10.6 p of dimethylamine dissolved in methanol was added and reacted. After gradually returning to room temperature, the solvent was removed and 3-(4-dimethylaminobutoxy)benzaldehyde phenylene! Lehydrazone 1
Measure 35f.

Dissolve 6 g of the obtained hydrazone in methanol and add potassium hydroxide solution while cooling. To this, 3.2f of 2-amino-4,5 dimethylthiazole hydrochloride and hydrochloric acid,
An aqueous solution of diazonium hydrochloride synthesized using nitrous acid was gradually added dropwise and stirred for 2 hours. Water was added to the reaction solution, cooled overnight, and then filtered to give 3-(4,5-dimethyl-2-
thiazolyl)-2-pheny)v-5-(3-(4-dimethylaminobutoxy)phenyl]formazan 4.69 is obtained. 25 fl of benzyl bromide) and 4 g of formazan are dissolved in tetrahydrofuran, copper oxide powder is added and heated. Reflux the crystals, dissolve them in methanol, filter, and concentrate the p solution under reduced pressure to extract the quaternary ammonium q2.
Get 59. 2 g of this quaternary ammonium salt is dissolved in methanol, and NB50.69111 is added thereto and stirred. Activated carbon is added to the reaction solution and filtered, and the filtrate is condensed to obtain the desired tetrazolium bromide.

Melting point 159-163°C (decomposition) a Elemental analysis 4L'h("i;) Cs+H*aNsO8
Br2. OCHN Theoretical value 51,82 5.33 11.70 Actual value 51,77 5.21 11.94'H-
NMR (δ, -DMSOIδP (TMS) 1.05-1
．． 72+m, 4H13,15~4.46(m, 18H)
4.91 (S, 2H) 6.85-7.46 (m,
'4H) 7.52-8.00 (rn, 5H) 8.
05-8.51 (m, 5H) IR (cm-') 2580 (-CI() 1600 (-C=N-)
1465(-CH11215+-COC-) 5
7ot-Q->Example 7 (Spectrophotometric determination method of dehydrogenase using the compound obtained in Example 6.) By the same measurement procedure as in Example 2, a good straight line passing through the origin as shown in Figure 3 was obtained. A sexual probe line was obtained. Note that the compound was used as a 00706% solution and the measurement was performed at 516 nm.

Example 8 (In general formula (1), Y is 2-hydroxypropylene, tty, R'' is 2-hydroxyethyl group,
X is a bromine atom, W is a hydrogen atom, 2 is 4.5-dimena IV
- A method for synthesizing a compound in which a 2-thiazolyl group is substituted with an li\-substituted r alkoxy group at the 4-position of a benzene ring. ) 4-oxirane methoxybenzaldehyde 25y obtained in Example 1
and 152 g of phenylhydrazine were added in methanol/M
Mix to synthesize 4-oxirane methoxybenzaldehyde phedi)V hydrazone 31y. 15 g of this hydrazone was dispersed in methanol and stirred, then 18 g of jetanolamine was added thereto, methanol was further added, and a potassium hydroxide solution was added while cooling. To this, 1M' of 2-amino-4,5-dimethylluteazo)V
Synthesis using l-lium 1. Add the diazonium hydrochloride water bath solution little by little and stir. Water was added to the reaction solution, allowed to stand overnight, and filtered to give 3-(4,5-dimethy)v-2-thiazolyl).
-2-phenyl-5-i4-[2-hydroxy-3-di[2-hydroxyethy/l/)aminopropoxy]pheny)vl formazan 18.31i+ is obtained 1, obtained formazan]l' is ethylene bromo Added to a mixed solvent of hydrin and tetrahydrofuran, stirred in S8 and condensed,
To it, add tetrahydrofuran to Jy + 11 -, 11 parts of holemazan, 4 parts of ammonium and 49.6 years of ammonium.
Ru. This 4g was dissolved in 8.59f of ammonium salt, and NB53.1& was added little by little and stirred.

Activated carbon was added to the reaction solution in a furnace of 711-1; the solution was condensed to give the desired tetrazolium bromide f4. 5°1 point 116-122°C Elemental analysis value ('-poly) CytHsaN. OJr.

CHN Theoretical value 48,37 5.41 10.45 Actual chain 48,74 5.56 10.02'H-NM
R(d, -DMS01δp(TMS) 3.26-4.6
3 (m, 24H17, 02-7.59fm, 4H)8.
00-8.451 rn, 5 HIIR(α-1) 3240(-OH) 2875(-CH) 1
600f-C=N-)1450(-CH)870(-Q
-1<4, brief explanation of the drawing
~ Figure 19 Figures 2 and 3 are respectively for Example 2. Example 5
492nm and 492nm according to Ober and Example 7, respectively.
The relationship between absorbance at 95 e'nm and 5]6 nm and LDH activity value is shown.

that's all Prisoner I L[)l-152 ÷ 1 shift

Claims (1)

[Claims] 1) A tetrazolium salt compound represented by the general formula. However, the meanings of the symbols in the formula are as follows. Be Y: A linear chain having 2 to 4 carbon atoms, or may have a side chain, and further has one hydroxyl group as necessary (-
alkylene group. R': Alkyl group having 1 to 4 carbon atoms or hydroxyethyl group. R1: 1 to 4 carbon atoms, optionally 1 to 2 hydroxyl groups,
or alkyl which may have one phenyl group/'! . X: chlorine atom, t or bromine atom. W: hydrogen atom or nitro group. Z: 4,5-dimethy)v-2-thiacyly/L' group when W is a hydrogen atom, 4-iodopheny)v group when W is a nitro group. 1 2) - Spectrophotometric determination of dehydrogenase using a tetrazolium salt compound represented by the general formula. However, the beauty of the symbols in the ceremony is as follows. Y: Straight chain with 2 to 4 carbon atoms, may have side chains. An alkylene group which may further have one hydroxyl group if necessary. R1: 7. Alkyl group having 1 to 4 carbon atoms, or hydroxyethyl RI: 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 or nitro group. z: When W is a hydrogen atom, 4,5-cymethy/l'-2-
thiacyly/I/ group, 4-iodophenyl)v group when W is a nitro group. 1st or 4th place.