JPS5939430B2 - Bistetrazolium 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 bistetrazolium salt compound and a method for spectrophotometric determination of dehydrogenase using the compound. Concerning the spectrophotometric determination method of

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

R1 is an alkyl group having 1 to 4 carbon atoms or a hydroxyethyl group.

R2: 1 to 4 carbon atoms, optionally 1 to 2 hydroxyl groups,
Or an alkyl group which may have one phenyl group.

R3: hydrogen atom or nitro group.

R4: Methoxy group: When R3 is a hydrogen atom, R4 may be a hydrogen atom.

X: chlorine atom or bromine atom.

In addition, the substitution position in the benzene ring of -0Y-N-R1 is the 3-position or the 3-position.

The compound having the above general formula (1) according to the present invention is a new substance that has not been described in any literature, and has the great feature that it can be used for the purpose of quantifying dehydrogenases because it accepts hydrogen and produces formazan.

That is, conventionally, a tetrazolium 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 is now widely used, especially in the field of clinical testing, and as a result, there are more and more opportunities to perform quantitative operations using automatic analyzers. It's coming. However, when known tetrazolium salts are used at that time, 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 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, glutamate dehydrogenase, etc., have been quantitatively analyzed using tetrazolium salt compounds. That is, the tetrazolium salt compound is
Formazan is produced 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 the heart muscle, liver, skeletal muscle, kidney, etc., and is associated with myocardial infarction, malignant tumors, liver disease, progressive muscle atrophy, and intravascular It is known that serum LDH activity increases markedly in cases of diseases such as hemolysis and megaloblastic anemia.
Therefore, clinically, by measuring LDH activity in the blood,
Extremely meaningful knowledge for diagnosis of the above-mentioned diseases can be obtained. Conventionally, as mentioned above, a tetrazolium salt compound, 3,3'-(3,37-dimethoxy4,4')
\biphenylylene)-bis[2-(4-nitrophenyl)-5-phenyl-2H tetrazolium salt compound] (hereinafter abbreviated as "nitro 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. Especially with the spread of automated analysis methods,
Although autoanalyzers are often used, the fatal drawback is that the formazan produced deposits in the tube of the analyzer, interfering 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 extremely undesirable as it also caused the problem to occur. 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, e.g. 3-(4-iodophenyl)-2-(4-nitrophenyl)-5phenyl-2H
Compounds such as tetrazolium chloride that slightly increase the water solubility of the produced formazan have also been used, but for the purpose of spectrophotometry, the water solubility of such formazan is still too low to be satisfactory for practical purposes. It's not a kimono. The present inventors have conducted various studies on compounds that produce formazan with good solubility and stable measurement values, and have found that the compound having the above general formula (1) is an excellent hydrogen acceptor for aqueous injection. 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 derivatives have problems such as being difficult to purify and, in the form of tetrazolium salts, having rather poor 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 has been established that allows stable measurement values to be obtained without worrying about precipitation. The compound having the general formula (1) according to the present invention can be produced by a conventional method.

For example, the corresponding 3 (or 4)-alkoxybenzaldehyde is first synthesized by reacting 3 (or 4)-hydroxybenzaldehyde with epichlorohydrin or a dihalogenated alkyl having 2 to 4 carbon atoms under basic conditions. . In addition, in the case of an alkoxybenzaldehyde compound having an oxirane ring, it may be reacted with hydrochloric acid or bromic acid under ice cooling to form a halohydrin compound. Now,
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 . . . rogene in its side chain may be reacted with a dialkylamine. Next, these hydrazone compounds are subjected to a coupling reaction with 0-dianisidine or dibenzidine αη zonium salt in the presence of a base to form a diformazane compound, and then diformazane having a tertiary amine in the side chain is coupled with an alkyl halide and an alkyl halide in the side chain. Formazan containing a halogen is reacted with a tertiary amine, and the corresponding 4
Synthesize ammonium salt compounds.

Next, diformazan having such a quaternary ammonium salt in its side chain was oxidized using an oxidizing agent such as n-butyl nitrite or N-bromosuccinimide (hereinafter abbreviated as "NBS").
Oxidation is performed using Z to obtain the desired bistetrazolium salt compound. These details will be described more specifically in the examples. Next, a method for spectrophotometric determination of dehydrogenation 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 monohydrochloride buffer, or the like. The hydrogen generated here is once accepted by nicotinamide adenine dinucleotide (hereinafter abbreviated as [NAD]), and then intermediate electron carriers such as diafluorase and phenazine methosulfate (hereinafter abbreviated as "PMS") Hydrogen is accepted by the bistetrazolium salt compound of the present invention via the body, thereby producing a 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 2-hydroxypropylene, R1 is ethyl group, R2 is 2-hydroxyethyl group, R
A method for synthesizing a compound in which 3 is a nitro group, R4 is a methoxy group, X is a chlorine atom, and a substituted alkoxy group is placed at the 4-position of a benzene ring.

) 4-Hydroxybenzaldehyde 1147 and epichlorohydrin 1707 are dissolved in tetrahydrofuran, a sodium hydroxide solution is added thereto, heated to reflux, and distilled under reduced pressure to obtain 4-oxiranemethoxybenzaldehyde 1007.
get. Next, this 4-oxirane methoxybenzaldehyde 507 and 4-nitrophenylhydrazine 43y are heated and mixed in methanol to synthesize 4-oxirane methoxybenzaldehyde-4-nitrophenylhydrazone 857. This hydrazone 40y was dispersed in methanol, diethylamine 307 was added, heated and concentrated under reduced pressure.
(2-Hydroxy-3-diethylaminopropoxy)benzaldehyde-4nitrophenylhydrazone 49y is obtained. The obtained hydrazone 427 is suspended in tetrahydrofuran together with O-dianisidine tetrazonium borofluoride salt 24y and cooled. Potassium hydroxide solution was gradually added dropwise to the mixture and stirred for 2 hours. Methanol was added to the reaction solution, filtered, and the 41y 1.'(3.3'-dimethoxy-
4,4'-biphenylylene)bis{5-(4-nitrophenyl)-3-[4(2-hydroxy-3-diethylaminopropoxy)phenyl]}diformazane is obtained. The obtained diformazan 407 is suspended in ethylene chlorohydrin, reacted while heating under reflux, and then concentrated. Tetrahydrofuran is added to the residue to obtain the quaternary ammonium salt 407 of the diformazan side chain. This quaternary ammonium salt 25
7 was dissolved in methanol, hydrochloric acid was added thereto, and while stirring at room temperature, butyl nitrite 15y was added thereto for reaction. The reaction solution is filtered and concentrated under reduced pressure to obtain the desired tetrazolium chloride 267. Melting point 167~17"C (
Decomposition) Elemental analysis value (%) C58H7ONl2Cl4, H2O Example 2 (Spectrophotometric determination of dehydrogenase using the compound obtained in Example 1.

) Glycine monolactate solution prewarmed to 37°C (glycine = 0.72%, dl-lactic acid = 0.42%, PH-9.
6) Add 0 to 82.6 mU/Ml of standard serum pre-warmed at the same temperature for 30 minutes to 4 ml and stir.

After 1 minute, 1 ml of 0.0710% aqueous solution of the compound obtained in Example 1 was added and stirred, and after another minute, NAD-PM
Add 0.1 ml of S solution (NAD-2%, PMSO.2%, bovine serum albumin fraction 5-0.15%, phosphate buffer of pH-7.4), stir, and heat to 37°C. Hold for 8 minutes.

Immediately add 1 ml of 0.1N hydrochloric acid, and measure the absorbance at 590 nm. 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 the serum could be accurately determined. Example 3 (Compound in which Y is propylene, R1 and R2 are ethyl groups, R3 is a nitro group, R4 is a methoxy group, X is a chlorine atom, and a substituted alkoxy group is substituted at the 3-position of the benzene ring in general formula (1)) synthesis method.

) 3-Hydroxybenzaldehyde 1227 and 1,3-
Dichloropropylene 5007 is dissolved in tetrahydrofuran, a sodium hydroxide solution is added thereto, the mixture is heated to reflux, and then distilled under reduced pressure to obtain 3-(3chloropropoxy)benzaldehyde 1187. Next, 63.57 of this 3-(3-chloropoxy)benzaldehyde and 467 of 4-nitrophenylhydrazine were heated and mixed in methanol,
3(3-chloropropoxy)benzaldehyde-4nitrophenylhydrazone 877 is synthesized. This hydrazone 53y and O-dianisidine tetrazonium borofluoride salt 307 are suspended in tetrahydrofuran and cooled. Potassium hydroxide solution was gradually added dropwise to this, and the mixture was stirred for 2 hours. Add methanol to the reaction solution, leave it overnight and filter it to obtain 1,11-(3,3'-dimethoxy-4,427).
4'-biphenylylene)-bis{5-(4-nitrophenyl)-3-[3-(3-chloropropoxy)phenyl]}diformazan is obtained. This formazan 30y and copper oxide powder were suspended in a mixed solvent of triethylamine and dioxane, and reacted while heating and refluxing. Methanol was added thereto, and the slurry was concentrated under reduced pressure to form a quaternary diformazane side chain. Ammonium salt compound 137 is obtained. 10 t of this quaternary ammonium salt is dissolved in methanol, hydrochloric acid is added thereto, and 12 y of butyl nitrite is added dropwise thereto while stirring at room temperature for reaction. Activated carbon is added to the reaction solution, filtered and concentrated under reduced pressure to obtain the desired tetrazolium chloride 87. Melting point 152-155°C (decomposed) Elemental analysis value (%) C53H7ONl2O8Cl4 Example 4 (In general formula (1), Y is 2-hydroxymethylethylene, R1 and R2 are propyl groups, R3 is a hydrogen atom,
A method for synthesizing a compound in which R4 is a methoxy group, X is a chlorine atom, and a substituted alkoxy group is substituted at the 4-position of the benzene ring.

) 4-oxirane methoxybenzaldehyde 23y obtained in Example 1 was slowly dropped into concentrated hydrochloric acid and stirred, then chloroform was added and a cold dilute aqueous sodium bicarbonate solution was added.
Next, the solvent was distilled off after washing with cold water, and the 4-(2-chloro-
3-Hydroxypropoxy)benzaldehyde 26y is obtained.

Next, 20y of 4(2-chloro-3-hydroxypropoxy)benzaldehyde and 107 phenylhydrazine were mixed in methanol while heating, and 4-(2-chloro3-hydroxypropoxy)benzaldehyde phenylhydrazone 2
Synthesize 57. This hydrazone 207 is dissolved in tetrahydrofuran and while cooling, a potassium hydroxide solution is added little by little, and then O-dianisidine tetrazonium borofluoride salt 157 is added little by little. After stirring for 2 hours, methanol was added to the reaction solution, allowed to stand overnight, and filtered thoroughly.
2f7 1・1′-(3・3′-dimethoxy-4・4′
biphenylylene)-bis{5-phenyl-3[4-(2
-chloro-3-hydroxypropoxy)phenyl]} diformazan is obtained.

This formazan 107 is suspended in a mixed solvent of tripropylamine and dioxane, heated and stirred, then methanol is added, filtered, and the slurry is concentrated under reduced pressure to obtain 6.37 of the quaternary ammonium salt of the diformazane side chain. . This quaternary ammonium salt 57 is dissolved in methanol, hydrochloric acid is added thereto, and 4.47 g of butyl nitrite is added thereto while stirring at room temperature for reaction. ．． The reaction solution is concentrated under reduced pressure to obtain 5.2y of the desired tetrazolium chloride. Melting point 126~13"C (
Decomposition) Elemental analysis value (%) C64H84NlOCl4O6, H2
Example 5 (Spectrophotometric determination of dehydrogenase using the compound obtained in Example 4).

) By the same measurement procedure as in Example 2, a calibration curve with good linearity passing through the origin was obtained, although the sensitivity was low, as shown in FIG.

The compound was measured as a 0.0698% solution at 5%
It was performed at 90 nm. Example 6 (In general formula (1), Y is 2-hydroxypropylene, R1 is a methyl group, R2 is a benzyl group, R3 and R4 are hydrogen atoms, X is a chlorine atom, and substituted alkoxy is at the 3-position of the benzene ring) Synthesis of substituted compounds.

) 3-Hydroxybenzaldehyde 867 and epichlorohydrin 1307 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-oxiranemethoxybenzaldehyde 75.
’F7 is obtained.

Next, 3-oxirane methoxybenzaldehyde 107
and phenylhydrazine 67 were heated and mixed in methanol, allowed to stand overnight, cooled, dimethylamine 127 was dissolved in methanol and added while stirring, allowed to react, and the temperature was gradually returned to room temperature, then the solvent was distilled off, and 3( 2-hydroxy-3
-dimethylaminopropoxy)benzaldehyde phenylhydrazone 177 is obtained. Obtained hydrazone 137
Dissolve in tetrahydrofuran, add potassium hydroxide solution and cool. A diazonium hydrochloride aqueous solution of dibenzidine was added little by little to this and stirred for 2 hours. Add 200ml of methanol to the reaction solution, leave it overnight, and filter thoroughly.
1,11-(4,47-biphenylylene)bis{5-phenyl-5-[3-(2-hydroxy-3-dimethylaminopropoxy)phenyl]}diformazan is obtained. The obtained formazan JMo V and copper oxide powder were suspended in a mixed solvent of benzyl chloride and dioxane and stirred, then methanol was added and the slurry was concentrated under reduced pressure to obtain the quaternary ammonium salt of the diformazan side chain. Get 5.5y. This quaternary ammonium salt 5y is dissolved in methanol, hydrochloric acid is added thereto, and while stirring, 4.87 g of butyl nitrite is added and reacted.
The reaction solution is thoroughly filtered and concentrated under reduced pressure to obtain the desired bistetrazolium chloride 5.17. Melting point 136-142
°C (decomposition) elemental analysis value (%) C62H64NlOO4C
l4 Example 7 (In general formula (1), Y is butylene,
A method for synthesizing a compound in which R1 is an ethyl group, R2 is a 2,3-dihydroxypropyl group, R3 is a hydrogen atom, R4 is a methoxy group, X is a chlorine atom, and a substituted alkoxy group is substituted at the 4-position of the benzene ring.

) Dissolve 84y of 4-hydroxybenzandehyde and 440y of 1,4-dichlorobutane in tetrahydrofuran,
A sodium hydroxide solution is added to this, heated to reflux, and then distilled under reduced pressure to obtain 4-(4-chlorobutoxy)benzaldehyde 627.

Next, this 4-(4-chlorobutoxy)benzaldehyde 2
87 and phenylhydrazine 14.27 are mixed under heating in methanol to synthesize 4-(4-chlorobutoxy)benzaldehyde phenylhydrazone 29y. This hydrazone 157 was dissolved in a mixed solvent of methanol and tetrahydrofuran Q, and diethylamine 21y was added while stirring, refluxed and concentrated under reduced pressure to produce 4-(4-diethylaminobutoxy).
Benzaldehyde phenylhydrazone 137 is obtained. The obtained hydrazone 13f and O-dianisidine tetrazonium borofluoride salt 8.57 are suspended in tetrahydrofuran and cooled. After gradually adding potassium hydroxide solution dropwise and stirring for 3 hours, methanol was added and left overnight.
3,3/-(1,11-dimethoxy-4,4!-biphenylylene)-bis{5-phenyl-3-[4-(4-diethylaminobutoxy)phenyl]
}Obtain diformazan. The resulting formazan 4.67 was dissolved in tetrahydrofuran, 3-chloro-1,2-dihydroxypropane 257 was added, and the mixture was heated to reflux. Methanol was added thereto, and the filtrate was concentrated under reduced pressure. 5.27 of grade ammonium salt is obtained. Dissolve 4.2y of this quaternary ammonium salt in methanol, add hydrochloric acid to it, and add 3.7y of butyl nitrite while stirring at room temperature.
Add 7 and react. After treating the reaction solution with activated carbon, it is filtered and concentrated under reduced pressure to obtain 4.87 g of the desired tetrazolium chloride. Melting point: 72-78℃ Elemental analysis value (
%)C62H8ONlOO8Cl4・2H20 Example
8 (Spectrophotometric determination of dehydrogenase using the compound obtained in Example 7).

) A calibration curve with good linearity passing through the origin as shown in FIG. 2 was obtained by the same measurement operation as in Example 2.

The compound was measured as a 0.0703% solution at 5%
It was performed at 82 nm. Example 9 (In general formula (1), Y is 2-hydroxyethylethylene, R1 and R2 are methyl groups, R3 is a nitro group, R4 is a methoxy group, X is a bromine atom, and the substituted alkoxy group is at the 3-position of the benzene ring) A method for synthesizing compounds substituted with .

) The 3-oxirane methoxybenzaldehyde 18y obtained in Example 6 was slowly dropped into bromic acid and stirred, then chloroform was added and the mixture was washed with a cold dilute aqueous sodium bicarbonate solution and then with cold water, and the solvent was distilled off. 3-(2-bromo-3-hydroxypropoxy)benzaldehyde21.
Get 57.

Next, this 3-(2-bromo-3-hydroxypropoxy)benzaldehyde 16y and 4-nitrophenylhydrazine 197 were heated and mixed in methanol, and 3-(2-bromo-3-hydroxypropoxy)benzaldehyde-
Synthesize 4-nitrophenylhydrazone 21y. This hydrazone 127 is O-dianisidine tetrazonium borofluoride salt 6.7? The mixture is suspended in tetrahydrofuran and cooled. Potassium hydroxide solution was gradually added dropwise to the mixture and stirred for 2.5 hours.

Add methanol 200111 to the reaction solution, filter it, and
．． 52-1,1'-(3,3'-dimethoxy-4,4'
-biphenylylene)-bis{5-(4-nitrophenyl)-3-[3-(2-furomo-3-hydroxypropoxy)phenyl]}diformazan is obtained. The obtained diformazan 87 and copper oxide powder are suspended in an aqueous trimethylamine solution and stirred, then methanol is added and the filtered slurry is concentrated under reduced pressure to obtain a quaternary ammonium salt of diformazan side chain 5.21. This quaternary ammonium salt 47 was dissolved in methanol, and NBSl. Add 67 little by little and react. The reaction solution was filtered and concentrated under reduced pressure to obtain 3.87 g of the desired tetrazolium bromide. Melting point 175
~180℃ (decomposition) Elemental analysis value (%) C52H58Nl2OlOBr4 (in general formula (1), Y is 2-hydroxypropylene, R
1, R2 is a 2-hydroxyethyl group, R3 is a nitro group,
Synthesis method of a compound in which R4 is a methoxy group, X is a chlorine atom, and a substituted alkoxy group is substituted at the 4-position of the benzene ring) Example 1
4-Oxiranmethoxybenzaldehyde obtained in
4-Nitrophenylhydrazone 207 is dissolved in tetrahydrofuran, O dianisidine tetrazonium borofluoride salt 127 is further added, and the mixture is cooled.

An aqueous potassium hydroxide solution was added dropwise to this, and the mixture was stirred for 2 hours. Add methanol to the reaction solution and filter it.
(3,3'-dimethoxy-4,4!-biphenylylene)
-bis{5(4-nitrophenyl)-3-(4-oxiranemethoxy)phenyl}formazan is obtained. The obtained diformazan is suspended in methanol in the presence of hydrochloric acid, triethanolamine is added thereto, and the suspension is heated for 20 hours. P
The liquid is concentrated and treated with tetrahydrofuran to obtain the quaternary ammonium salt 167 of the diformazane side chain. Quaternary ammonium salt 107 is dissolved in methanol, hydrochloric acid is added thereto, and n-butyl nitrite is added and reacted with stirring at room temperature.

The reaction solution is thoroughly filtered and concentrated to obtain the desired tetrazolium chloride 9.5. Melting point 183-19 "C (decomposition) Elemental analysis value (%) C58H7, Nl2Ol6Cl4

[Brief explanation of the drawing]

1, 2 and 3 are Example 2 and Example 5, respectively.
and 590 nm, respectively, according to Example 8.
It is a chart showing the relationship between absorbance at 590 nm and 82 nm and LDH activity value.

Claims (1)

[Claims] 1. A bistetrazolium salt compound represented by the general formula. 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. R^3: Hydrogen atom or nitro group. R^4: Methoxy group; When R^3 is a hydrogen atom, R
^4 may be a hydrogen atom. X: chlorine atom or bromine atom. In addition, ▲There are mathematical formulas, chemical formulas, tables, etc. ▼The substitution position on the benzene ring is the 3-position or the 3-position. 2 Spectrophotometric determination of dehydrogenase using a bistetrazolium salt compound represented by the general formula. However, the meaning in the formula is as follows. Y: Number of carbon atoms is 2
~4 straight chain type or alkylene group which may have a side chain and further may have one hydroxyl group as required. 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. R^3: Hydrogen atom or nitro group. R^4: Methoxy group; When R^3 is a hydrogen atom*
▲There are mathematical formulas, chemical formulas, tables, etc.▼Regarding the tetrazolium salt compound represented by ▼ and the method for spectrophotometric determination of dehydrogenase using that compound. However, the meanings of the symbols in the formula are as follows. Y: Linear type with 2 to 4 carbon atoms, or may have a side chain, and if necessary, has one hydroxyl group ▲ Numerical formula, chemical formula, table, etc. are available ▼・・・・・・( I) *R^4 may be a hydrogen atom. X: chlorine atom or bromine atom. In addition, the substitution position in the benzene ring of ▲, which includes mathematical formulas, chemical formulas, tables, etc., is the 3rd or 4th position.