JPH0321016B2 - - Google Patents

Description

Detailed Description of the Invention [] Background Technical Field of the Invention The present invention relates to an aniline derivative having two reactive groups capable of reacting with a group having active hydrogen, particularly a hydroxyl group and an amino group, and its production and use. . More specifically, the present invention relates to aniline derivatives having groups with activated halogen atoms as reactive groups, their production and their uses. Recently, immunoassay has begun to be applied to the analysis of plant components, and examples of its application to alkaloids and glycosides have already been reported (Zenk, MH et al.
Planta medica, 41 (1), p1 (1981)). This method is
The basic idea is to bind a protein to an alkaloid or glycoside through an appropriate group, such as a hydroxyl group, and perform analysis using an antigen-antibody reaction using the resulting sugar-protein crosslinked protein as an antigen. That is. However, most applications to glycosides are based on aglycones, which are sugar-free structures. The main reason for this is
This is due to the fact that it is easy to bind to the carrier protein during immunogen preparation, and that the aglycone portion has most of the physiological activity of the target glycoside. However, in immunoassay methods using aglycone as a control, It has not always been possible to apply this method to searching for drug distribution, metabolism, etc. in plants. For this purpose, crosslinking between glycosides and proteins is required. In addition, it is desirable that this crosslinking reaction be mild so that the structure of the glycoside itself will not be significantly changed by the crosslinking site, and that the crosslinking reaction can be applied to all glycosides. reagent,
In particular, the development of a compound that selectively binds to a C-6 hydroxyl group and an amino group is desired. It goes without saying that if such a crosslinking reagent is developed, it will become possible to crosslink glycosides with enzymes such as peroxidase, and the establishment of enzyme immunoassays will become extremely easy. Also,
This cross-linking reagent is also expected to be applied to research on the synthesis of artificial glycoproteins, which has been attracting attention recently, and as a reagent for immobilizing lidand in affinity chromatography. Prior Art Various studies have been conducted on the reactivity of the four hydroxyl groups of glucosides. Alkylation, acetylation (F.Bottiuo, etal.Ann.Chim.
(Rome) 1972, 62 , 782-8) C-6
The reactivity towards the C-2, C-3 and C-4 positions is the highest, but all reactions are non-selective. In addition, p-toluenesulfonyl chloride is known as a selective modification reagent for the C-6 position of glucosides (Cramer, F. etal. Chem. Ber. 1959,
92, 384-391), but it has not been applied to crosslinking reagents. The following methods are known as methods for crosslinking sugars and proteins. (1)Periodic acid oxidation method Nakane, PKetal: J.Histochem.Cytochem.
22, 1084 (1974) and Nakane.PKetal:
Method Enz. 37 , 133 (1975) (2) A method in which an amino group is introduced into the C-1 position and then crosslinked with a carboxyl group of a protein using EDC. (3) Direct reduction method with sodium borohydride Karol, MHetal: Proc.Nat.Acad.Sci.USA
57, 713 (1967) and Erlanger, BFetal:
Acta Endocrinol.Suppl. 168 , 206 (1972) However, (1) and (3) change the structure of the sugar, and (2) is a crosslink at the C-1 position, so it selectively connects the C-6 position of the glucoside. The drawback is that it cannot be applied to cross-linking reactions with proteins. Thus, until now, few effective methods have been developed for selectively crosslinking specific structural parts with proteins and other target substances without changing the structure of the sugar moieties. Therefore, for the above purpose, there is a need for a bivalent or bireactive crosslinking reagent that acts as a "linker" that selectively binds to sugars and proteins. [] Summary of the Invention From this point of view, the present inventors have considered the application to all glycosides and other physiologically active substances containing sugar, and have developed a method for connecting proteins through the C-6 moiety of sugar. We have developed a bivalent cross-linking reagent that enables cross-linking of glucoside (α-methyl-
We investigated the cross-linking reaction between D(+)-glucoside) and BSA (bovine serum albumin). In principle, the present invention attempts to utilize the nucleophilic substitution reaction of a primary hydroxyl group of a sugar to an acid halide and the nucleophilic substitution reaction of an amino group or imidazole nitrogen of a protein to a carbonylmethyl halide. The aim is to use aniline derivatives with active groups for each of these reactions as a crosslinking reagent. That is, the aniline derivative according to the present invention is represented by the following formula. Here, the substituents have the following meanings. R ¹ = -CH ₂ X ¹ or -( ^CH ₂ ) _o -COCH ₂ X ¹ (n is an integer ^of 2 or more) R ² = ^-SO ₂ ² = each halogen atom other than fluorine The method for producing an aniline derivative represented by the following formula according to the present invention is an aniline derivative consisting of aniline sulfonic acid or aniline carboxylic acid and a functional derivative of the sulfonic acid or carboxylic acid moiety. N ^- acylation of the acid derivative of aniline with an acyl halide of the ^formula It is characterized by being attached to . Here, the substituents have the following meanings. R ¹ =-CH ₂ X ¹ or =- ⁽ CH ₂ ) _o -COCH ₂
is an integer ^of 2 or more) R ² = -SO ₂ ^{X 2} or ^{-COX 2} The method for producing aniline derivatives is to convert aniline into the formula
N by acyl halide represented by X ³ COR ¹
-acylation and halogenation of the acid with a compound represented by the formula X ⁴ R ³ . Here, the substituents have the following meanings. R ¹ = −CH ₂ X ^{1 ,} or − ⁽ CH ₂ ) _o −COCH ₂ X ¹ (n is an integer of 2 or ^more ) R ³ = _−SO ² = Each halogen atom other than fluorine X ⁴ = A halogen other than fluorine that is the same as or different from X ¹ to X ³ or a hydroxyl group. Effects According to the present invention, C-6 of glycoside (glucoside)
We were able to obtain a bivalent reagent that can selectively and reactivity crosslink the ε-amino group of lysine and the imidazole ring nitrogen of histidine in proteins. Glucoside and the acid halide group (R ²
or R ³ ), the benzene ring of this reagent plays a very large role. That is, the benzene ring reacts with C-6, which is the most reactive among the four hydroxyl groups of glucoside, and at the same time reacts with C-2, C-3, and C-4 due to its steric hindrance.
It is interfering with the reaction with the position. In addition, acid halide and terminal carbonylmethyl halide group (-
The difference in reactivity with COCH ₂ X ² ) also indicates the specificity of this reagent. That is, the hydroxyl group of glucoside can only be attacked by acid halide, while the carbonylalkyl halide group remains stable without any change until the subsequent reaction with protein. From this point of view, this reagent is a useful and excellent crosslinking reagent. When applied to immunoassays, this reagent enables cross-linking of carrier proteins and enzymes for enzyme immunoassays with glycosides without changing the structure of the sugar moiety, making it easy to synthesize immunogens. . Furthermore, since the reagent itself also serves as a spacer, it is expected that highly specific antibodies that recognize the entire hapten molecule can be produced. According to this reagent, it is possible to cross-link a specific site, that is, the C-6 position, with a protein without changing the structure of the sugar moiety, and therefore, it is possible to specify the structure of the produced glycoprotein. Benefits are obtained during glycoprotein synthesis. Cross-linking with enzymes facilitates the establishment of enzyme assay systems, facilitates the structure of cross-linked hapten molecules, and does not change the structure of cross-linked hapten molecules, resulting in improved binding ability with antibodies and associated It is also expected to improve analytical sensitivity. In applications to affinity chromatography, it is believed that all crosslinking is possible under mild conditions for supports and ligands having primary hydroxyl groups and amino groups. As described above, the aniline derivative according to the present invention can be said to be a selective reagent that has a wide range of applications and is highly expected to be used in the future. [] Specific description of the invention 1 Compound 1 Definition Compound according to the present invention (hereinafter referred to as the present compound)
is a novel aniline derivative represented by the following formula. Here, the substituents have the following meanings. _R1 = _-CH2X1 ^, or -( _CH2 ) _{o -COCH2X1} (n is an integer of 2 or more, preferably 10 or ^less , particularly preferably 5 or less) ^R2 = ^-SO2 X ² or COX ² X ¹ and X ² = each a halogen atom other than fluorine. As is clear from the notation of R ² in the above formula,
The substituent R ² is o-,
It may be in either the m- or p-position. Representative examples of the present compounds are those in which R ¹ is -CH ₂ X ¹ , those in which X ¹ is bromine, and those in which X ² is chlorine. Some specific examples of the present compound are as described below in connection with the production method. 2.Synthesis The present compound is synthesized by any purposeful method, including introduction of the desired substituent to aniline, which is the base compound, and conversion of the already introduced precursor substituent to the desired substituent. be able to. One such method involves the use of acid derivatives of aniline, i.e., aniline sulfonic acid (i.e., aminobenzenesulfonic acid) or aniline carboxylic acid (i.e., aminobenzoic acid), or the sulfonic or carboxylic acid moieties of these acids. Functional derivatives such as salts (e.g. alkali metal salts, amine salts, etc.), acid halides (e.g. chloride), etc., of the formula X ³ COR ¹ (X ³ is X ¹ to ^{X 2}
a halogen atom other than fluorine that is the same or different from
R ¹ is as described above) and, if the acid derivative of aniline used is not an acid halide of aniline sulfonic acid or aniline carboxylic acid, conversion of the acid derivative to the acid halide. It consists of things. The conversion of acid derivatives of aniline to acid halides can be carried out according to conventional methods, for example, when the acid derivative is a free acid, by reaction with a customary acid halogenating agent, such as thionyl chloride. . Specific examples of acyl halogenides ^of the formula X ³ COR ^{1 that} are N-acylating agents are X ³ COCH ₂ X ¹ and X ³ CO(CH ₂ ⁾ _o COCH ₂ n is as above). Specific examples of X ¹ are bromine, specific examples of X ³ are bromine or chlorine, and specific examples of n are 2-5. The N-acylation reaction is preferably carried out in a solvent or dispersion medium. As the reaction solvent or dispersion medium, water is suitable for acid derivatives of aniline, but in order to minimize the contact between the N-acylation reagent and water and suppress the formation of byproducts such as haloacetic acid, bromoacetic acid, etc. In addition, in order to dissolve the reactive components in the organic layer and facilitate extraction and separation, the acid derivative of aniline is dissolved in the aqueous layer and the N-acylating agent (e.g., bromoacetyl bromide) is dissolved in the organic layer. A two-layer system is preferred.
Examples of the organic layer include dichloromethane. The reaction temperature is about 5 to 35°C. If the acid derivative of aniline used is not an acid halide, the N-acylated intermediate obtained as described above is treated with a conventional acid halide reagent such as a thionyl halide or a phosphorus halide, such as thionyl chloride, pentachloride, etc. By reacting with phosphorus, phosphorus tribromide, etc., the acid part becomes an acid halide group (R ² ).
The desired compound is obtained. This reaction can be carried out at a temperature of about 80 to 90°C in an organic solvent or dispersion medium, if necessary. One of the other methods of preparing the present compounds consists in subjecting aniline to N-acylation with an acyl halide of the formula X ³ COR ¹ and said acid halide with a compound of the formula X ⁴ R ³ (R ³ is -SO ₂ X ² , other substituents are as described above). Although either N-acylation or acid halogenation may be carried out first, it is preferable to carry out the former first. For details of the N-acylation step, reference may be made to what has been described for the N-acylation of acid derivatives of aniline.
In this case as well, a two-layer reaction medium consisting of an aqueous layer and an organic layer is preferred. The acid halogenation step may also be any suitable one. The reaction is usually carried out at a temperature of about 50 to 70°C in the presence or absence of a solvent. In addition to these methods, the present compound can be synthesized by any suitable method. For example, to synthesize a compound in which the substituent R ² is -COX ² ,
If necessary, the desired -
It can also be converted to COX ² . 3 Specific examples of compounds Compounds (1), (2), (3) are specific examples of intermediate products.
and (12), and compound (4) as an example of the present compound,
(5) and (13) are shown in the formula below. 2 Utilization of the present compound Since the present compound has two active halogen-containing groups that can react with a hydroxyl group and an amino group, it can be used for various purposes by utilizing its reactivity. 1 Crosslinking Reagent One example of such a use is as a crosslinking reagent for crosslinking the sugar moiety of a glycoside and a protein, as described above. The crosslinking reaction between the glycoside and the protein may be carried out stepwise or simultaneously, but it is preferable to carry out the crosslinking reaction stepwise. Furthermore, even when the reaction is carried out in stages, it is preferable to carry out the reaction with the glycoside first. For the former, α-methyl-D(+)-glucoside (α-
MeGlu) and the latter, bovine blood albumin (BSA), are explained below as representative examples. 2. Reaction with glycoside The present compound and α-MeGlu are reacted in a suitable solvent to selectively form an ester bond at the C-6 position of α-MeGlu. The solvent is preferably one that can completely dissolve the participating compounds, especially both starting compounds, and neutralize the by-product HCl. Examples of such solvents include 2,6-lutidine-dioxane. Even in the case of this solvent, it is necessary to increase the solubility by adding hexamethylphosphoric triamide (HMPT) or the like. Lutidine used in this system also has the advantage of suppressing the formation of quaternary salts of nitrogen due to the steric hindrance of its methyl group. In addition, compounds (6) and (7) are shown in the following formulas as specific examples. 3 Reaction with protein BSA used as an example of protein contains
59 lysines (Lys) and 17 histidines (His)
The compound forms a bond with each amino acid or imidazole ring nitrogen.
Reaction analysis is performed by determining the number of reactions from the results of amino acid analysis. (1) Synthesis The present compound (in this analysis, intermediates and derivatives for group R ² are also included) and BSA are reacted at any temperature and at any molar ratio. The BSA fraction and unreacted substances are separated. In this case, it is preferable to use a Sephadex G-25 column. In addition, as specific examples of reaction products, compound (8),
(9), (10) and (11) are shown in the formula below. (2) Results of measuring the number of reactions Hydrolyze the BSA fraction with acid, perform amino acid analysis using a conventional method, and calculate the number of reactions based on the reduced number of Lys and His. The reaction results of the above-mentioned compounds (1), (2), (6), and (7) with BSA are shown in Tables 1 and 2 below (for details of the experiment, refer to the following experimental example). In Table 1, an increase in the number of reactions is observed as the molar ratio of compound (2) to BSA increases and as the BSA concentration increases. The observed change in the number of reactions of compound (5) is considered to be dependent on the solubility of compound (5). Also, from Table 2, the number of reactions of compounds (6) and (7) bonded to sugar does not depend much on the reaction temperature,
Although there was little change as the concentration of BSA increased, a significant increase was observed when the molar ratio of compound (6) or (7) to BSA was increased. 3 Experimental Example Experimental Example 1 10g (57.52mmol) of sulfanilic acid at 0.805N
- Dissolve in 150 ml of sodium hydroxide aqueous solution and add 5.51 ml of bromoacetyl bromide while stirring on ice.
(63.28mmol) is added. The reaction solution is kept at pH 8 by adding a concentrated aqueous sodium hydroxide solution, and then allowed to react for 2 hours with ice-cooling and stirring, and further allowed to react overnight at 4°C. When the reaction solution is freeze-dried, Compound (1) is obtained almost quantitatively as a white powder. Identification IR1175cm ^-1 (-SO ₃ Na) ¹ H-NMR (D ₂ O, 60MHz) δ (ppm) 4.67 (s, 2H, -CH ₂ -) 8.19, 8.38 (ABq, 4H, -Ph, J _AB = 8.0
Hz) Experimental example 2 10g (72.92mmol) of p-aminobenzoic acid
Dissolve in 1400ml of 0.1094N-sodium hydroxide aqueous solution and add 6.99ml of bromoacetyl bromide to this.
(16.19g, 80.22mmol) and 360ml of dichloromethane
Add all at once and allow to react at room temperature for 15 minutes while stirring vigorously. Add 73 ml of 1N hydrochloric acid to the reaction solution, collect the resulting precipitate, and wash it three times with 500 ml of distilled water.
After drying, compound (2) is obtained as a white powder. Yield 11.6g (yield 85.6%) Identification IR 1680cm ^-1 (C=O) Mass M ⁺ =257, M ⁺ +2 = 259 ¹ H-NMR (DMSO-d ₆ , 60MHz) δ (ppm) 4.05 ( s, 2H, −CH ₂ −) 6.30−7.43 (br.1H.−NH−) 7.70, 7.88 (ABq.4H.−Ph, J _AB =9.0
Hz) 10.60 (brs.1H, -COOH) Experimental example 3 10g (9.78ml, 107.39mmol) of aniline
Dissolve in 1400ml of 0.0844N-sodium hydroxide aqueous solution, and add 10.29ml of bromoacetyl bromide to this.
(23.84g, 118.13mmol) and dichloromethane
Add 500 ml of the mixture at once and react for 15 minutes at room temperature with vigorous stirring. After separating the dichloromethane layer, it is washed twice with 300 ml of distilled water, concentrated under reduced pressure, and the solvent is distilled off to obtain compound (3) as a white powder. Yield 19.5g (yield 84.8%) Identification IR 1655cm ^-1 (C=O) ¹ H-NMR (DMSO-d ₆ , 60MHz) δ (ppm) 4.00 (s, 2H, -CH ₂
−) 7.00−7.68 (m, 5H, −Ph) 10.72 (br.1H, −NH−) Experimental example 4 10 g of compound (1) and 50 ml of thionyl chloride
(68.84mmol) is added. Then in an oil bath
The mixture is heated to 80-90°C and the reaction is carried out under reflux for 4 hours. After completion of the reaction, unreacted thionyl chloride is distilled off under reduced pressure at 40° C., and then thionyl chloride is completely removed under reduced pressure using a vacuum pump to obtain Compound (4) as a yellow powder almost quantitatively. Identification IR1360cm ^-1 (-SO ₂ Cl) Mass M ⁺ = 311, M ⁺ +2 = 313, M ⁺ +4 = 315 Experimental example 5 Add 50% of thionyl chloride to 10g (38.91mmol) of compound (2)
Add ml (68.84mmol). Then in an oil bath
The mixture is heated to 80-90°C and the reaction is carried out under reflux for 4 hours.
After completion of the reaction, unreacted thionyl chloride is distilled off under reduced pressure at 40° C., and thionyl chloride is completely removed under reduced pressure using a vacuum pump to obtain Compound (5) as a white powder almost quantitatively. Identification IR1736cm ^-1 , 1680cm ^-1 (C=O) Experimental Example 6 Under ice-cooling and stirring, 40.82g (23.29g) of chlorinated sulfonic acid
ml, 350.33 mmol) to compound (3) 15 g (70.06 mmol)
Add. After that, heat it to 60℃ in an oil bath, and
Allow time to react. Pour the syrupy product into 500 ml of ice water while stirring. Compound (4) is obtained by filtering the resulting precipitate, washing with water, and drying. Yield 18.2g (yield 82.9%) Identification IR1660cm ^-1 (C=O) 1165cm ^-1 (-SO ₂ Cl) Mass M ⁺ =311, M ⁺ +2 = 313, M ⁺ +4 = 315 ¹ H-NMR (DMSO−d ₆ , 60MHz) δ (ppm) 4.28 (s, 2H, −CH ₂ −) 7.55 (s, 4H, −Ph−) 10.43 (br, 1H, −NH−) Experimental example 7 Glucoside (α− Add 1 ml of lutidine to 100 mg (0.515 mmol) of methyl-D(+)-glucoside,
Furthermore, by adding a few drops of hexamethylphosphoric triamide, the glucoside is completely dissolved. Dissolve 323 mg of compound (4) in 1 ml of dioxane,
Add the above solution while stirring on ice. After another hour, 300 mg of compound (4) dissolved in 1 ml of dioxane.
and react overnight at 4°C. After confirming the product by TLC (silica gel, CHCl ₃ -MeOH8:2), 30 ml of distilled water was added to the reaction solution, and the mixture was extracted and washed four times with 50 ml of n-hexane to remove lutidine from the aqueous layer. The remaining product was transferred to a silica gel column ("Wakogel C-200", 2 x 25 cm, mobile phase CHCl ₃
-MeOH7.5:2.5). After evaporating the solvent, it is dissolved in distilled water and freeze-dried to obtain compound (6) as a white powder. Yield 92 mg (yield 38%) Identification ¹ H-NMR (D ₂ O, 100MHz) δ (ppm) 3.30 (s, 3H, −OCH ₃ ) 4.30 (s, 2H, −CH ₂ ) 4.40−3.32 ( m, 7H, sugarH) 7.85, 7.70 (ABq, 4H, -Ph, J _AB = 9.0Hz) ¹³ C-NMR (D ₂ O, 100MHz) δ (ppm) δ (ppm) C-1 49.1 C-7 78.9 2 61.1 8 105.2 3 75.1 9 126.7 4 75.1 10 135.1 5 75.8 11 135.4 6 76.9 12 148.5 C-13 173.9 FD-Mass M ⁺ = 470, M ⁺ + 2 = 472 Experimental example 8 To 150 mg (0.772 mmol) of glucoside (α-methyl-D(+)-glucoside), add 1.5 ml of lutidine, and then add several doses of hexamethylphosphoric triamide. Completely dissolve the glucosides by adding dropwise. Add 400 mg of compound (5) and react at 4°C overnight. TLC (silica gel, _CHCl3 -MeOH8:
After confirming the product by 2), add distilled water to the reaction solution.
25 ml of the solution was added, and lutidine was removed from the aqueous layer by extraction and washing six times with 35 ml of n-hexane. Thereafter, the product is extracted six times with 35 ml of ethyl acetate, and the solvent is distilled off under reduced pressure at below 40°C. The remaining product was transferred to a silica gel column ("Wakogel C-200", 2 x 25 cm, mobile phase CHCl ₃
-MeOH8:2), and after evaporation of the solvent, it is dissolved in distilled water, frozen, and dried to yield almost quantitative compound (7) as a white powder. Identification ¹ H-NMR (DMSO-d ₆ , 100MHz) δ (ppm) 3.26 (s, 3H, -OCH ₃ ) 4.30 (s, 2H, -CH ₂ ) 4.56-3.30 (m, 7H, sugarH) 7.92, 7.74 (ABq, 4H, -Ph, (J _AB = 9.0Hz) ¹³ C-NMR (DMSO-d ₆ , 100MHz) δ (ppm) δ (ppm) C-1 43.5 C-8 99.6 2 54.3 9 118.7 3 64.1 10 124.6 4 69.5 11 130.2 5 70.3 12 142.7 6 71.7 13 165.1 7 73.1 FD-Mass M ⁺ = 433, M ⁺ + 2 = 435 Experimental example 9 Dissolve 50 mg of BSA (bovine serum albumin) in 1 ml of sodium borate buffer (0.01M-Na ₂ B ₄ O ₇ , PH 9.18) and store at room temperature (23 6.87 mg (30.00 times equivalent) of compound (1) was added to this, and the mixture was stirred vigorously at room temperature (23 °C).
℃) overnight. After the reaction is complete, the reaction solution
Take 50 μ (approx. 2.5 mg of BSA) and add Cephadex G
-25 column 1 x 30 cm, mobile phase: 10% acetic acid) to separate the BSA fraction and unreacted compound (1).
Take 0.2 mg of BSA from the BSA fraction and add concentrated hydrochloric acid to make a 6N-hydrochloric acid solution. After degassing, seal the tube under reduced pressure and hydrolyze at 110°C for 24 hours. After concentrating the hydrolyzate, it was dissolved in 350μ of 0.02N hydrochloric acid and
Perform amino acid analysis using an 835 amino acid analyzer,
Find the number of decreases in Lys (lysine) and His (histidine). The reaction results are shown in Table 1. Experimental Example 10-14 Compound (1) or (2) was prepared by changing the molar ratio or (and) molar concentration (volume of solvent) under the reaction conditions.
The reaction between and BSA is carried out in the same manner as in Experimental Example 9 above. The results of the reaction are shown in Table 1. Experimental Example 15 50 mg of BSA (bovine serum albumin) was dissolved in 1 ml of sodium borate buffer (0.01M-Na ₂ B ₄ O ₇ , PH9.18), stirred vigorously under ice cooling, and 10 mg of compound (6) was added to this.
(29.33 times equivalent) and react at 4°C overnight. After the reaction is complete, add 50μ of reaction solution (approximately 2.5mg of BSA)
Take a Sephadex G-25 column (1 x 30 cm,
Mobile phase: 10% acetic acid) to separate the BSA fraction and unreacted compound (6). 0.2 mg equivalent than BSA fraction
Take BSA and add concentrated hydrochloric acid to make a 6N-hydrochloric acid solution.
After degassing, seal the tube under reduced pressure and hydrolyze at 110°C for 24 hours. Hydrolyzate is concentrated with 0.02N-HCl 350μ
The solution is dissolved in water, subjected to amino acid analysis using a Hitachi 835 amino acid analyzer, and the number of decreases in Lys (lysine) and His (histidine) is determined. The reaction results are shown in Table 2. Experimental Examples 16 and 17 Among the reaction conditions, the reaction between compound (6) and BSA was carried out in the same manner as in Experimental Example 15, by changing the reaction temperature, solvent, or molar ratio. The reaction results are shown in Table 2. Experimental Example 18 50 mg of BSA (bovine blood albumin) was dissolved in 1 ml of sodium borate buffer (0.01M-Na ₂ B ₄ O ₇ , PH9.18), stirred vigorously under ice cooling, and compound (7) 10
mg (31.87 times equivalent) and reacted at 4°C overnight. After the reaction is complete, add 50μ of reaction solution (approximately 2.5mg of BSA)
Take a Sephadex G-25 column (1 x 30 cm,
Mobile phase: 10% acetic acid) to separate the BSA fraction and unreacted compound (7). Take 0.2 mg of BSA from the BSA fraction and add concentrated hydrochloric acid to make a 6N-hydrochloric acid solution. After degassing, seal the tube under reduced pressure and hydrolyze at 110°C for 24 hours. The hydrolyzate is concentrated with 0.02N hydrochloric acid.
Dissolve in 350 ml and perform amino acid analysis using a Hitachi 835 amino acid analyzer to detect Lys (lysine) and His.
Find the number of decreases in (histidine). The reaction results are shown in Table 2. Experimental Examples 19-21 Among the reaction conditions, the reaction between compound (7) and BSA is carried out in the same manner as in Experimental Example 18, by changing the reaction temperature, solvent and/or molar ratio. The reaction results are shown in Table 2. Experimental Example 22 10 g (72.92 mmol) of m-aminobenzoic acid was dissolved in 100 ml of diethyl ether, and lutidine was added to it.
Add 18.58ml (17.19g, 160.42mmol). 5-
Bromlevrinyl chloride 17.13g (80.21mmol)
was dissolved in 100 ml of diethyl ether, added to the above solution under ice-cooling and stirring, and allowed to react for 1 hour.
After the reaction is completed, unreacted m-aminobenzoic acid, generated HCl, and lutidine are removed by washing the reaction solution three times with 200 ml of 0.2N HCl. The ether was concentrated under reduced pressure and analyzed by TLC (silica gel, CHCl ₃
-MeOH8:2) After confirming the product,
Silica gel column (“Wakogel C-200”, 2x
25 cm, using mobile phase _CHCl3 -MeOH8:2).
Separate and purify. After distilling off the solvent from the fraction containing the product, it is dissolved in distilled water and freeze-dried to obtain compound (12) as a white powder. Yield 18.3g (yield 80.2%) Identification IR 1692cm ^-1 (C=0) Mass M ⁺ =314, M ⁺ +2 = 316 Experimental example 23 Add 50% of thionyl chloride to 10g (31.82mmol) of compound (12)
Add ml (68.84mmol). Thereafter, the mixture is heated to 80 to 90°C in an oil bath, and the reaction is carried out under reflux for 4 hours. After the reaction is complete, remove unreacted thionyl chloride at 40°C.
When the thionyl chloride is completely removed under reduced pressure using a vacuum pump, yellow powder compound (13) is obtained. Yield 1.6g (yield 15%) Identification IR 1690, 1745cm ^-1 (C=0) Mass M ⁺ =333, M ⁺ +2 = 335, M ⁺ +4 = 337 [Table] [Table] [Table]

Claims (1)

[Claims] 1. An aniline derivative represented by the following formula. Here, the substituents have the following meanings. R ¹ = −CH ₂ X ¹ , or −(CH ₂ ⁾ _o −COCH ₂ H ¹ (where n is an integer ^of 2 or more) ^{R 2} = ^SO ₂ , halogen atoms other than fluorine. 2. A compound according ^to claim 1, wherein ^R2 is _SO2X2 . 3. The compound according to claim 1, wherein R ² is SOX ² . 4. The compound according to claim 1, which is represented by the following formula. 5. The compound according to claim 1, which is represented by the following formula. 6. The compound according to claims 1 to 5, wherein X ¹ is bromine and X ² is chlorine. 7 N-acylation of acid derivatives of aniline consisting of aniline sulfonic acid or aniline carboxylic acid and functional derivatives of the sulfonic acid or carboxylic acid moiety with an acyl halide of the formula X ³ COR ¹ and the use of the aniline A method for producing an aniline derivative represented by the following formula, which comprises converting the acid derivative into an acid halide when the acid derivative is not an acid halide of aniline sulfonic acid or aniline carboxylic acid. Here, the substituents have the following meanings. R ¹ = −CH ₂ X, or −(CH ₂ ⁾ _o −COCH ₂ H ¹ (where n ^is an integer ^{greater than} or equal to ² ) R ² = SO ₂ Each is a halogen atom other than fluorine. 8. The method according to claim 7, wherein the acid derivative of aniline is a free acid or a salt. 9. The method according to claims 7 to 8, wherein the acid halogenation is performed after the N-acylation. 10 N-acylation of aniline with an acyl halide of formula X ³ COR ¹ and
A method for producing an aniline derivative represented by the following formula, which comprises subjecting the acid to halogenation with a compound represented by X ⁴ R ₃ . Here, the substituents have the following meanings. R ¹ = −CH ₂ X ¹ , or −(CH ₂ ⁾ _o −COCH ₂ H ¹ (where n is an integer of ² or more ⁾ R ₃ = _−SO ² Halogen atoms other than fluorine. 11. The method according to claim 10, wherein ^X4 = a halogen other than fluorine that is the same as or different from ^X1 to ^X3 , or a hydroxyl group 11 N-acylation is performed and then the acid halogenation is performed.