JPH05170752A - 3,7-bis(disubstituted)phenothiazine derivative and its production - Google Patents

Abstract

PURPOSE:To obtain a new compound useful as a photosensitive dye capable of readily modifying an antibody protein, etc. CONSTITUTION:A compound expressed by formula I (R1 to R4 are 1-6C alkyl and at least one thereof has a group reactive with amino, thiol or carboxyl as a substituent group; X is group capable of forming counter ion with phenazathionium), e.g. 3-[N-methyl-N-(4-succinimidooxycarbonylbutyl) amino]-7- dimethylaminophenazathionium chloride. The compound expressed by formula I is obtained by releasing Y in a compound expressed by formula II [at least one of R1' to R4' is group expressed by the formula (CH2)m-CO-Y and others are 1-6C alkyl; Y is protecting group; (m) is 1-6; X<-> is counter ion of the phenazathionium] and then introducing a group reactive with the amino, thiol or carboxyl group thereinto. Thereby, modification of an antibody protein, etc., with a photosensitive dye can be facilitated to prevent a protein-dye conjugate from settling and nonsecifically bonding to solid surfaces which are problems in the modification.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel phenothiazine derivative and a method for producing the same. The compound of the present invention is a derivative capable of binding methylene blue, which is one of the photosensitizing dyes, to antibody proteins and the like.
It is a compound that is expected to be applied to immunoassays that utilize chemoluminescence and todydynamic therapy.

[0002]

2. Description of the Related Art As a practical example utilizing the oxidative characteristic reaction of light-sensitive dyes, disinfection and sterilization of water with methylene blue is well known. To date, extensive research has been conducted on the photoexcitatory activity of photosensitizing dyes such as xanthines such as rose bengal, fluorescein, eosin, and erythrosine, and phenothiazines represented by methylene blue and porphyrins. There is a trend toward increasing applications in the field of diagnostic agents. Of these photo-sensitive dyes, methylene blue has an unusually strong photo-oxidation effect and is excellent in that it absorbs long-wavelength light (670 nm) that easily penetrates biological tissues. Excitation activity of methylene blue by light is due to foot (mechanism of photosensitized oxidation) [Foote (Mechanismsof Ph
autosensitized oxidation, 1
November 29, 968, Volume 162, 963-970.
It is believed that it consists mainly of two reactions, as reported by [Page. That is, in the type I photoexcitation reaction, the excited dye directly reacts with the substrate, and in the case of type II, the excited triplet dye first reacts with molecular oxygen to generate singlet oxygen. It is thought that it is generated and then the substrate is oxidized, and which route it takes is determined by the concentrations of the dye, dissolved oxygen and the substrate.

Recently, among these photo-sensitive dyes, the use of porphyrins for cancer treatment has been reported [Analytical Chemistry (Analyticala).
Chemistry), Vol. 61, Dec. 15, 1989, pp. 1367-1375]. It is said that porphyrins are injected into or near the cancer tissue and excited by receiving light energy from the outside of the body to generate singlet oxygen, resulting in the death of cancer cells. One of the important characteristics of such dyes is that they can absorb long-wavelength light (> 650 nm) that can penetrate biological tissues. As an attempt to treat such cancer, an improved method utilizing the specificity of the antigen-antibody reaction has recently been reported. That is, it is a method of specifically attacking the cancer cell itself, which is an antigen, by binding a light-sensitive porphyrin dye to an antibody against cancer (JO 2059-58).
5-A; European Patent No. 252683). However,
Most of these photosensitive dyes are not easy to bind to antibody proteins. Thus, if readily available highly reactive photosensitizing dyes that are easy to couple with proteins and that can absorb long wavelength light (> 650 nm) can be expected to bring significant improvements in this field.

Another important use of photosensitive dyes is in the field of diagnostics. For example, the photosensitive dyes herein may be antibodies, haptens or nucleic acids (DN
(A, RNA) used for labeling probes. The obtained labeled antibody, hapten or nucleic acid probe is used in the process of clinical chemical analysis.
It is used for the generation of a signal indicating an The signal is derived from a labeled photosensitive dye, and colorimetric, fluorescent, or chemiluminescent is typical. However, this method has hardly been used because a highly reactive derivative of a photosensitive dye capable of covalently binding a protein to a hapten or a nucleic acid has been difficult to obtain.

[0005]

The ideal derivative of a photosensitive dye is to have an active functional group that can easily react with a protein, hapten or nucleic acid under normal reaction conditions. One of these active functional groups is a succinimide ester group capable of reacting with an amino group of a protein or nucleic acid, and the other is a maleimide group capable of reacting with a thiol group of a protein. It is difficult to directly introduce such a functional group into a methylene blue dye, which is a fact that, although methylene blue has been the subject of much research for over 50 years, a modifiable derivative of protein or nucleic acid has not yet been found. It is also obvious that it is not. When a low molecular weight aromatic compound such as a dye is bound to a protein or a hapten (eg, thyroid hormone), precipitation of a dye-catalyst (protein-dye conjugate) and nonspecific binding to a solid surface are often caused. It becomes a problem. These two problems are caused by the hydrophobicity of the dye in an aqueous solution, resulting in a decrease in the solubility of the dye-catalyzed label and non-specific adsorption of the protein, hapten or nucleic acid on the surface of the solid phase. Thus, if it is possible to improve such instability of the dye-catalyzed label or find a method for preventing non-specific adsorption, it becomes possible to greatly improve the conventional technique.

[0006]

In view of the above-mentioned problems, the present inventors have synthesized various reactive derivatives of methylene blue, and as a result of their studies, have found that a hydrophilic group such as a carboxyl group is introduced into the methylene blue skeleton. The inventors have found that it is effective in improving the instability of the dye-catalyzed labeled product, and completed the present invention.

That is, the present invention provides (1) the general formula:

[0008]

[Chemical 15]

[Wherein R ₁ , R ₂ , R ₃ and R ₄ are each a C _1-6 alkyl group, at least one of which is a group which reacts with an amino group, a thiol group or a carboxyl group. And X ⁻ represents a counter ion of phenazathionium].

(2) General formula:

[0011]

[Chemical 16]

[In the formula, at least one of R ₁ ', R ₂ ', R ₃ 'and R ₄ ' is a group represented by the formula:-(CH ₂ ) _m -CO-Y, and the other is C _{1-. 6 represents} an alkyl group, Y represents a leaving group,
m represents an integer of 1 to 6 and X ⁻ represents a counter ion of phenazathionium].

(3) General formula:

[0014]

[Chemical 17]

[In the formula, at least one of R ₁ ', R ₂ ', R ₃ 'and R ₄ ' is a group represented by the formula:-(CH ₂ ) _m- CO-Y, and the other is C _{1-. 6 represents} an alkyl group, Y represents a leaving group,
m represents an integer of 1 to 6] an indamine thiosulfonic acid derivative represented by:

(4) General formula:

[0017]

[Chemical 18]

[Wherein R ₁ ″ represents a C _1-6 alkyl group,
Y represents a leaving group, m represents an integer of 1 to 6] 2-amino-5-disubstituted aminophenylthiosulfonic acid derivative;

[0019]

[Chemical 19]

[Wherein at least one of R ₁ ', R ₂ ' and R ₃ 'is a group represented by the formula:-(CH ₂ ) _m- CO-Y, and the other is a C _1-6 alkyl group. , Y represents a leaving group, and m is 1 to
Represents an integer of 6] (6) General formula:

[0021]

[Chemical 20]

[Wherein at least one of R ₁ ′, R ₂ ′, R ₃ ′ and R ₄ ′ is a group represented by the formula: — (CH ₂ ) _m —CO—Y, and the other is C _{1- 6 represents} an alkyl group, Y represents a leaving group,
m represents an integer of 1 to 6, X ⁻ represents a counter ion of phenazathionium, and Y of the phenothiazine derivative represented by
Is removed, and then a group that reacts with an amino group, a thiol group or a carboxyl group is introduced:

[0023]

[Chemical 21]

[Wherein R ₁ , R ₂ , R ₃ and R ₄ are each a C _1-6 alkyl group, at least one of which is a substituent group which reacts with an amino group, a thiol group or a carboxyl group. And X ⁻ represents a counter ion of phenazathionium]. (7) General formula:

[0025]

[Chemical formula 22]

[Wherein at least one of R ₁ ′, R ₂ ′, R ₃ ′ and R ₄ ′ is a group represented by the formula: — (CH ₂ ) _m —CO—Y, and the other is C _{1-. 6 represents} an alkyl group, Y represents a leaving group,
m represents an integer of 1 to 6], an indamine thiosulfonic acid derivative represented by the general formula: is oxidatively ring-closed with an inorganic oxidizing agent in water or an organic solvent or a mixed solvent thereof.

[0027]

[Chemical formula 23]

[Wherein at least one of R ₁ ', R ₂ ', R ₃ 'and R ₄ ' is a group represented by the formula:-(CH ₂ ) _m- CO-Y, and the other is C _{1-. 6 represents} an alkyl group, Y represents a leaving group,
m represents an integer of 1 to 6 and X ⁻ represents a counter ion of phenazathionium] to obtain a phenothiazine derivative, the Y of which is eliminated, and then the phenothiazine derivative is reacted with an amino group, a thiol group or a carboxyl group. A general formula characterized by the introduction of a group

[0029]

[Chemical formula 24]

[Wherein R ₁ , R ₂ , R ₃ and R ₄ are each a C _1-6 alkyl group, at least one of which is a group which reacts with an amino group, a thiol group or a carboxyl group as a substituent. And X ⁻ represents a counter ion of phenazathionium]. (8) General formula:

[0031]

[Chemical 25]

[Wherein B represents a succinimidooxycarbonyl group or a maleimido group and m represents an integer of 1 to 6], and a triethylamine salt of an acidic amino acid is reacted with the succinimide ester derivative. General formula to do:

[0033]

[Chemical formula 26]

[Wherein B represents a succinimidooxycarbonyl group or a maleimido group, m represents an integer of 1 to 6 and n represents 1 or 2]; (9) General formula:

[0035]

[Chemical 27]

[Wherein R ₁ , R ₂ , R ₃ and R ₄ are the same or different and each is a C _1-6 alkyl group, at least one of which reacts with an amino group, a thiol group or a carboxyl group. Group having a group as a substituent, X ⁻ represents a counter ion of phenazathionium], and a labeled product obtained by coupling a phenothiazine derivative represented by (10) A general formula in which an antigen or antibody is coupled to an antigen or antibody through its amino group, thiol group or carboxy group:

[0037]

[Chemical 28]

[Wherein R ₁ , R ₂ , R ₃ and R ₄ are the same or different and each is a C _1-6 alkyl group, at least one of which reacts with an amino group, a thiol group or a carboxyl group. A phenothiazine derivative having a group as a substituent and X ⁻ represents a phenazathionium counter ion].

The groups reactive with an amino group, a thiol group or a carboxyl group in R _{1 to} R ₄ of the compound of the general formula (I) are a protein, a hapten, a peptide, a nucleic acid, an amino acid to be subjected to an immunological test or the like. Although it is not particularly limited, it may be any group capable of reacting with etc., but as a typical example, (1) a group which reacts with an amino group is a carboxyl group, a halogenocarbonyl group (eg, a chlorocarbonyl group) , Azidocarbonyl group, cyanocarbonyl group, alkoxycarbonyloxycarbonyl group (eg, ethoxycarbonyloxycarbonyl group, isobutoxycarbonyloxycarbonyl group), substituted phenoxycarbonyl group (eg, p-nitrophenoxycarbonyl group, 2,4-dinitro Phenoxycarbonyl group, pentachlorophenoxycarbonyl group, , 4,5-trichloro phenoxycarbonyl group, pentafluoro phenoxycarbonyl group),
1-imidazolylcarbonyl group, 1-benzotriazolyloxycarbonyl group, 5-norbornene-2,3-dicarboximidooxycarbonyl group, succinimidooxycarbonyl group, maleimidooxycarbonyl group,
Aspartic acid residue and glutamic acid residue; (2) Examples of the group that reacts with a thiol group include a maleimide group;
(3) Examples of the group that reacts with a carboxyl group include an aminoalkylcarbamoyl group (eg, aminoethylcarbamoyl group, aminodecylcarbamoyl group) and the like.

In the present invention, for that purpose, it is necessary that such a reactive group is contained in at least one of the R _{1 to} R ₄ groups of the general formula (I). Further, the compound of the general formula (I) is preferably hydrophilic for the purpose,
The number of such reactive groups can be appropriately selected depending on the combination of _{1 to} R ₄ groups. The C _1-6 alkyl groups represented by R _{1 to} R ₄ are the same or different and each is a linear or branched C _1-6 alkyl group, for example, methyl, ethyl, propyl, isopropyl, butyl. , Sec-butyl,
Examples thereof include tert-butyl, pentyl, isopentyl, neopentyl, hexyl, and isohexyl, with methyl and ethyl being particularly preferred from the viewpoint of structural similarity to methylene blue and hydrophilicity. The C _1-6 alkyl group in R ₁ ′ to R ₄ ′ and other groups in the present specification also shows the same group.

As the group capable of forming a counter ion with phenazathionium represented by X in the above general formulas (I) and (II), halogen (eg chlorine, bromine, iodine, fluorine), hydroxyl, perchlorine Acid, sulfuric acid, nitric acid, trifluoro acid, borofluoric acid and the like can be mentioned. The general formula
(II), (III), (IV) and formula :-( CH ₂ in (V)) _m -
The leaving group represented by Y in CO-Y may be a leaving group usually used in the fields of peptide synthesis, β-lactam antibiotics and the like, and examples thereof include methoxy, ethoxy, propoxy, sec-butoxy and tert-butoxy. 1 to 4 carbon atoms
And lower alkoxy groups and aralkyloxy groups such as benzyloxy, p-nitrobenzyloxy, p-methylthiobenzyloxy, diphenylmethyloxy and 4-pyridylmethoxy, and 1-phenoxyethoxy groups.

The compound represented by the general formula (I) is, for example, Y of a phenothiazine derivative represented by the general formula (II).
Is removed, and then a group reactive with an amino group, a thiol group or a carboxyl group is introduced.
Specifically, for example, it can be manufactured according to the steps of the chart 1 shown below.

Chart 1 [In Chart 1, m is an integer of 1 to 6, n is 1 or 2,
X ⁻ represents a counter ion of phenazathionium. ]

[0044]

[Chemical 29]

[In the formula, at least one of R ₁ ′, R ₂ ′, R ₃ ′ and R ₄ ′ is a group represented by the formula: — (CH ₂ ) m-CO—Y, and the other is C _{1- 6 represents} an alkyl group, and Y represents a leaving group. ]

[0046]

[Chemical 30]

[Wherein at least one of R ₅ ′, R ₆ ′, R ₇ ′ and R ₈ ′ is a group represented by the formula: — (CH ₂ ) m-COOH, and the other is C _1-6 alkyl. Indicates a group. ]

[0048]

[Chemical 31]

[Wherein R ₉ ', R ₁₀ ', R ₁₁ 'and R ₁₂ '
At least one of the groups is a group represented by the formula:-(CH ₂ ) m-COA, the other is a C _1-6 alkyl group, and -COA is a group which reacts with an amino group. ]

[0050]

[Chemical 32]

[Wherein R ₁₃ ', R ₁₄ ', R ₁₅ 'and R ₁₆ '
At least one is a group represented by the formula:-(CH ₂ ) m-CONH-CH (COOH)-(CH ₂ ) n-COOH, and the other is a C _1-6 alkyl group _or- (CH ₂ ) m.
-COA (-COA is as defined above)]

That is, (1) Among the phenothiazine derivatives represented by the general formula (I), the compounds having a group which reacts with an amino group in R _{1 to} R ₄ are the compounds represented by the general formula (II). The carboxylic acid derivative obtained by subjecting it to hydrolysis, catalytic reduction or acid decomposition reaction (acidolysis) is halogenated, azidated,
It can be produced by a known method involving mixed acid anhydride formation or active esterification reaction. In the chart, the group that reacts with the amino group represented by —COA is the _one described above as the group that reacts with the amino group represented by R _{1 to} R ₄ (however, an aspartic acid residue and a glutamic acid residue). Excluding).

The ester hydrolysis of the compound represented by the general formula (II) can be usually carried out in a 1N solution of mineral acid or caustic at a reaction temperature of 20 to 30 ° C. for a required time of 20 minutes to 48 hours. . However, under such conditions, the decomposition of the phenothiazine skeleton also proceeds at the same time, so the yield is generally low, and this tendency is particularly remarkable under alkaline conditions. When the leaving group in the compound (II) is an aralkyloxy group such as benzyloxy, p-nitrobenzyloxy, 4-pyridylmethoxy and diphenylmethyloxy, these can be removed by catalytic reduction. The reaction is usually carried out by treating with a palladium catalyst in a hydrogen stream at room temperature and atmospheric pressure in an inert solvent. At this time, the phenothiazine skeleton is also reduced along with elimination of the ester to form a leuco-type phenothiazine derivative, which is rapidly air-oxidized to the original phenothiazine.

On the other hand, an alkoxy group such as tert-butoxy, an aralkyloxy group such as p-methylthiobenzyloxy or diphenylmethyloxy, or a 1-phenoxyethoxy group can be easily treated by treatment in formic acid or trifluoroacetic acid. Can be desorbed. The reaction is usually completed in 30 to 60 minutes at a reaction temperature of 0 to 25 ° C. The organic acids can be used alone or as a mixture with an inert solvent. Among the leaving groups represented by the compound (II), the tert-butoxy group is most preferable in view of easiness of removal and purification after elimination. That is, when the leaving group is a tert-butoxy group, it can be used in the next step simply by concentrating to dryness after completion of the reaction. If further purification is required, silica gel column chromatography (developing solvent MeCN / H ₂ O / 1N HCl = 4
0: 5: 1) is possible. The carboxylic acid derivative thus obtained is then coupled with a compound having an amino group to be introduced into a reactive derivative such as an acid chloride, an acid azide, a mixed acid anhydride or an active ester.

A more specific description will be given below. Acid chloride method Thionyl chloride was added to the carboxylic acid derivative in an inert solvent such as dichloromethane, chloroform, carbon tetrachloride or benzene.
Chlorides such as oxalyl chloride, trichloromethylchloroformate (diphosgene), bis-trichloromethylcarbonate (triphosgene) are allowed to act at 0 to 25 ° C,
After forming the corresponding acid chloride, it is subjected to a coupling reaction with an amino compound in the presence of an organic tertiary amine such as triethylamine, diisopropylethylamine, pyridine and 4-dimethylaminopyridine.

The hydrazide derivative derived from the acid azide method carboxylic acid derivative is described by Curtius, Chem. Ber. , 3
5 , 3226 (1902)], the acid azide compound produced after neutralization by the action of sodium nitrite in an acidic solution is subjected to solvent extraction for coupling reaction, or the neutralized reaction solution is subjected to in situ reaction. It can also be subjected to a coupling reaction. This azidation reaction can also be carried out in a non-aqueous solvent using alkyl nitrite instead of sodium nitrite [J. Rudinger (J.
Rudinger) et al., Collect. Czech.
Chem. Commun. , 26 , 2333 (196
1)]. Furthermore, this derivative is a diphenylphosphoric acid azide (DPPA) [T. Shioir (T. Shioir
i) et al. Am. Chem. Soc. , 94 , 620
3 (1972)].

Mixed Acid Anhydride Method As an acid component which forms a mixed acid anhydride with a carboxylic acid derivative, a carbonic acid monoalkyl ester is preferable, and ethoxycarbonyl chloride [tee. T. Wieland et al.
Ann. Chem. , 572 , 190 (1951); RA Boissonna (RA Boissonna).
s), Helv. Chim. Acta. , 34 , 874
(1951)] or isobutyloxycarbonyl chloride [JR Vaugha (JR Vaugha
n) et al. Am. Chem. Soc. , 74 , 676
(1952)] at −15 to −5 ° C. and can be directly reacted with an amino compound.

Active ester method A phenyl ester having an electron-withdrawing substituent is known as one of active esters used for peptide synthesis, but it can also be applied to the present carboxylic acid derivative. That is, esters with p-nitrophenol, 2,4-dinitrophenol, pentachlorophenol, 2,4,5-trichlorophenol and pentafluorophenol are convenient.

On the other hand, O-
Acyl hydroxylamine-based esters are known.
For example, 1-hydroxybenzotriazole [W. Koenig et al., Chem. Ber. ,
103 , 788 (1970)], N-hydroxysuccinimide [G.W. Anderson (GW).
Anderson) et al. Am. Chem. So
c. , 85 , 3039 (1963); ibid, 86 , 183.
9 (1964)], N-hydroxymaleimide (the above N
-Hydroxysuccinimide) or N-hydroxy-5-norbornene-2,3-dicarboximide [M.Fujino]
Chem. Pharm. Bull. , 22 , 185
7 (1974)] and the like. Also, N
-Hydroxyphthalimide [G.H.L. Nefkeus et al., J. Am.
Chem. Soc. , 83 , 1263 (1961)].
Esters of and can be similarly used.

These active ester derivatives were prepared by adding N, N-dicyclohexylcarbodiimide (DC
C) or a condensing agent such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (WSC: water-soluble carbodiimide). In addition to the above, from the reaction with 1,1′-carbonyldiimidazole (CDI), 1-acylimidazole [S. Staab, Angew. Che
m. Internat. Edit. , 1 , 351 (19
62)] is also cyanide diethylphosphate (DEPC)
From the reaction with acyl cyanide [T. Shioiri et al., Tetrahedron
Lett. , 1973 , 1595] and the like, and any of the reactive derivatives can be used in the direct reaction with the amino derivative.

Among the phenothiazine derivatives represented by the general formula (I) thus obtained, the compounds having a group capable of reacting with an amino group in R _{1 to} R ₄ can be prepared by appropriately selecting the coupling method described above. Peptide bonds can be formed with amino compounds such as haptens, nucleic acids or amino acids. The condensation method is not particularly limited and can be appropriately selected depending on the properties of the amino compound. In particular, the azide method, the mixed acid anhydride method, and the active ester method are preferable as the coupling method that can be performed in an aqueous solution, but considering the stability of the reactive derivative, the high reactivity, and the ease of purification after the reaction, , O-acylhydroxylamine-based esters, especially with N-hydroxysuccinimide are most preferred.

The succinimide ester can be prepared, for example, in an anhydrous aprotic solvent such as acetonitrile, dimethylacetamide or dimethylformamide,
In the presence of 2-3 equivalents of N-hydroxysuccinimide,
Equivalent amount of dicyclohexylcarbodiimide (DCC) or 1-ethyl-3- (3-dimethylaminopropyl)
Reacting carbodiimide hydrochloride (WSC) (20-40
By carrying out the reaction at 1 ° C. for 1 to 48 hours), a monoester body, or in the case of a dicarboxylic acid, a mixture with a diester body containing a monoester body as a main product is obtained. When the intended diester form is used, it is preferable to use 3 to 6 equivalents of the reaction reagent.

In these esterification reactions,
The succinimide esterification is carried out using N, N′-disuccinimide carbonate (DSC) [H.Ogura (H.Og
ura) et al., Tetrahedron Lett. , 1
979 , 4745] or N, N'-disuccinimide oxalate (DSO) [H. Ogur (H. Ogur
a) et al., Tetrahedron Lett. , 24 ,
It is preferable to use an active ester of N-hydroxysuccinimide such as 4451 (1983)]. Usually, in the case of monoesterification in the presence of twice the amount of the active ester in an inert solvent such as acetonitrile, tetrahydrofuran, dioxane or acetone, for example, in the presence of an organic base such as pyridine or triethylamine, 1. It is preferable to use 5 to 3 equivalents, in the case of diesterification, 3 to 6 equivalents of the active ester.

The reaction is usually completed in 1 to 3 hours at 20 to 30 ° C. When the starting material is a dicarboxylic acid derivative,
It is often difficult to produce only the monoester form, but these separations are performed using, for example, Sephadex (S
It is possible by column chromatography with ephadex) (registered trademark) LH-20 (developing solvent: acetonitrile). The obtained succinimide ester is reacted with a triethylamine salt of aspartic acid or glutamic acid (MeCN / H ₂ O = 3: 1, 25 ° C., 10 minutes) to give a compound represented by the general formula (VII) or the general formula (VII ′ in the chart). The compound shown by these can be manufactured.

(2) Of the phenothiazine derivatives represented by the general formula (I), compounds having a group capable of reacting with a thiol group in R _{1 to} R ₄ , that is, a maleimide group, such as R ₄
Is a maleimidoalkyl group (general formula (VI),
(VII) in which B is a maleimido group) is a compound of the general formula (V), that is, a compound of the general formula (II) in which R ₄ 'is a maleimidoalkyl group. _A compound in which 4'is a maleimidoalkyl group can be produced by treating in the same manner as in the case of a compound having a group which reacts with an amino group. In addition, (3) among the compounds represented by the general formula (I), R _{1 to} R ₄
The phenothiazine derivative having a group that reacts with a carboxyl group is obtained by adding the reactive derivative of phenothiazinecarboxylic acid shown in (1) to the formula H ₂ N- (CH ₂ ) p-NH ₂ [wherein p is 2 to The monoacylaminoalkylamine obtained by reacting the diamino compound represented by the formula [10] is further subjected to reaction with another carboxyl group or a reactive derivative thereof.

The compound represented by the general formula (II), which is a starting material for producing the compound represented by the general formula (I), can be prepared by a known method of synthesizing methylene blue according to the steps shown in Chart 2. A. Bernthen
sen), Annalender Chemie, 25
1 , 1, (1888); I. Tomioka (I. Tomi
oka), J. Chem. Ind. Tokyo Japan
an, 20 , I-2 (1917)].

[0067]

[Chemical 33]

[In the formula, at least one of R ₁ ′, R ₂ ′, R ₃ ′ and R ₄ ′ is a group represented by the formula: — (CH ₂ ) m—CO—Y, and the other is C _{1-. 6 represents} an alkyl group, Y represents a leaving group,
X ⁻ represents a counter ion of phenazathionium. However, the synthetic intermediate of the compound of the present invention is far more lipophilic than methylene blue, and satisfactory results cannot be obtained by simple application of the methylene blue synthetic method in which the reaction solvent is all water. Thus, the reaction solvent is preferably water or an organic solvent, especially acetonitrile or acetone, and may be used in a mixed state thereof, if necessary. Further, as the oxidizing agent in each oxidation step, it is possible to use an inorganic oxidizing agent such as dichromate, ferric chloride, manganese dioxide which has been conventionally used, in a water-containing organic solvent which is a feature of the present invention. Dichromate or manganese dioxide is preferred as the oxidant used in the oxidation reaction in step 1.

Each step will be described more specifically.
N, N-dialkyl-p-phenylenediamine obtained from a monoalkylaniline (wherein alkyl means methyl or ethyl) by a conventional method by N-alkylation and nitrosation, followed by reduction with water or acetone, In a water-containing organic solvent such as acetonitrile, 1.
In the presence of 2-3.5 equivalents of sodium thiosulfate, 1.0-3.0 equivalents of aluminum sulfate and 8.0-12.0 equivalents of acid (eg hydrochloric acid, sulfuric acid or potassium acid sulfate).
0.3-4.0 equivalents, preferably 0.3-2.0 equivalents of dichromate are used at a reaction temperature of 0-40 ° C, preferably 5-25 ° C.
The compound represented by the general formula (IV) is obtained by reacting with. Also, this compound is 3.0 instead of dichromate.
It can also be produced using 1 to 10 equivalents of manganese dioxide.

The compound represented by the general formula (IV) is treated with an equivalent amount of the compound represented by the formula (V) in water, aqueous acetone or aqueous acetonitrile solution.
0.1) in the presence of a compound of formula III) and 1.0 to 3.0 equivalents of an acid (eg hydrochloric acid, sulfuric acid or potassium hydrogen sulfate).
When 6-3.0 equivalents, preferably 0.6-1.5 equivalents of dichromate are reacted at 0-40 ° C., preferably 5-25 ° C., the indamine compound represented by the general formula (III) is used. An osulfonic acid derivative is obtained. Also, the oxidizer of this reaction is 3.0 to
Substituted by 10.0 equivalents of manganese dioxide.

The compound represented by the general formula (II) is obtained by oxidatively ring-closing the indamine thiosulfonic acid derivative represented by the general formula (III) with an inorganic oxidizing agent in water or an organic solvent or a mixed solvent thereof. It is obtained by doing. More specifically, the compound represented by the general formula (III), water, acetone, acetonitrile or a water-containing solvent thereof,
It is represented by the general formula (II) when treated at 20 to 100 ° C. in the presence of 5.0 to 20.0 equivalents, preferably 10.0 to 15.0 equivalents of manganese dioxide, and more preferably a catalytic amount of copper sulfate. A new methylene blue derivative is obtained. In addition, during the steps shown in Chart 2, without isolating the intermediates (IV) and (III), the required reaction reagent was added to the slurry for each step to make the compound (IV) consistent. ((I
I) can also be produced.

The compounds of the present invention can be coupled with various proteins such as antibodies, antibody fragments and avidin (basic glycoprotein). When a photosensitive dye derivative is used for the treatment of cancer, it is easily taken up by cancer cells, for example, transferrin protein [transferferr].
in protein (iron transport
It is necessary to select a protein or biomolecule required for the growth of cancer cells, such as protein).

The time required for coupling with the protein is 1 minute to 24 hours, preferably 4 to 12 hours. The temperature at the time of coupling is preferably 1 to 80 ° C. Coupling at a high temperature is usually suitable at 1 to 24 ° C. because the reaction is promoted but protein denaturation also occurs. P at the time of coupling
H is 4 to 10, but preferably 6 to 8. The coupling is usually carried out in an aqueous solution, but a solvent such as alcohol, acetone or dimethylformamide, which has no strong nucleophilicity and does not react with the active reactive derivative, can be used. Similarly, any buffer salt for pH control may be used as long as it does not react with the reactive derivative. For example, tris (hydroxymethyl) aminoethane and the like are unsuitable as buffer agents, and phosphates and the like are suitable.

Essentially all proteins have a primary amino group and are capable of reacting with the compounds of this invention. In reacting the compound of the present invention having an amino group-reactive group with a compound having an amino group such as protein and hapten,
When the group that reacts with the amino group is a carboxyl group, it is convenient to carry out in the presence of a condensing agent. Examples of such condensing agents include DCC and WSC. There should be no impurities having a mercapto group other than the reduced protein cysteine in the protein solution at the time of coupling with the maleimide derivative. Immune test and photodynamic treatment of cancer
Antibody proteins are particularly recommended for use in herpay.
The molar ratio of the reactive photosensitive dye derivative to the protein at the time of coupling is larger than 1, and preferably 10 to 300. After coupling the reactive dye derivative to the protein, the produced dye-catalyzed label can be separated from the unreacted dye by, for example, gel filtration chromatography. The prepared dye catalyst label can be stored in an aqueous solution for a long time. However, high pH (10 and above)
The dye is unstable in the area and cannot be stored.

For example, coupling of a reactive dye derivative to a low molecular weight hapten such as a drug or a derivative thereof, a hormone or a nucleic acid can be carried out under the same conditions as described above, and the produced labeled product can be purified. It can be carried out by means such as chromatography, which can distinguish the product from the unreacted raw material. The use of dye-catalyzed labels in immunoassays involves oxidative chemistry by irradiating the dye with an appropriate wavelength of light that is excited and combining the product from the excited dye with a sensing reagent, such as luminol. This can be done by detecting luminescence. When the dye-catalyzed labeled product is used for photodynamic treatment of cancer, semiconductor laser light (670 nm) is particularly preferable because the light spectrum of the light source and the absorption spectrum of the dye are well matched and easily available.

[0076]

EXAMPLES The present invention will be described in more detail below with reference to reference examples and examples, but the present invention is not limited thereto. Reference Example 1 N- (3-ethoxycarbonylpropyl) -N-methyl-
Ethyl p-phenylenediamine 4- (N-methylanilino) butyrate (JCSPerkin
I, 1972, 1803) (22 g) was dissolved by adding concentrated hydrochloric acid (60 ml) under ice-cooling, and a solution of sodium nitrite (8 g) in water (10 ml) was added dropwise at 0 to 4 ° C. with vigorous stirring. After stirring at the same temperature for 30 minutes, 30 g of zinc dust was added at 10 ° C or lower, and the mixture was further stirred at 15 ° C for 30 minutes. Zinc was filtered off, the filtrate was made alkaline with sodium hydrogen carbonate and then extracted with ether. The ether layer was dried over anhydrous sodium sulfate, concentrated, and the residue was subjected to silica gel column chromatography and developed with ethyl acetate / hexane = 1: 1 to give 9 g of N-ethoxycarbonylpropyl-N-.
Methyl-p-phenylenediamine was obtained. NMR (CDCl ₃ ) δ: 1.21 (3H, t, J = 7Hz), 1.
84 (2H, quintet, J = 7Hz), 2.30 (2H, t, J =
7Hz), 2.80 (3H, s), 3.1-3.5 (2H, br.),
3.20 (2H, t, J = 7Hz), 4.10 (2H, q, J = 7H
z), 6.63 (4H, s). IR (neat) ν: 3450, 3350, 1730, 152
0, 1370, 1265, 1175, 815 cm ^-1 .

Reference Example 2 Ethyl 5- (N-methylanilino) valerate 16 g of N-methylaniline, ethyl 5-bromovalerate 3
After stirring 0.2 g of the mixture at 110 ° C. for 22 hours,
The mixture was made alkaline with aqueous sodium hydrogen carbonate, extracted with ethyl acetate, and dried over anhydrous magnesium sulfate. After concentration, the residue was distilled under reduced pressure to obtain ethyl 5- (N-methylanilino) valerate (28.6 g). bp: 124-128 ° C / 0.
4 mmHg NMR (CDCl ₃ ) δ: 1.25 (3H, t, J = 7.2Hz),
1.66 (4H, m), 2.33 (2H, t, J = 8Hz), 2.9
1 (3H, s), 3.32 (2H, t, J = 7Hz), 4.12 (2
H, q, J = 7.2 Hz), 6.67 (3 H, m), 7.22 (2 H,
m).

Reference Example 3 Ethyl 6- (N-methylanilino) hexanoate A mixture of 25 g of N-methylaniline and 50 g of ethyl 6-bromohexanoate was stirred at 120 ° C. for 24 hours. After cooling, the mixture was made alkaline with aqueous sodium hydrogen carbonate, extracted with ethyl acetate, washed with brine, and dried over anhydrous sodium sulfate. After concentrating under reduced pressure, the residue was distilled to obtain 40.5 g of ethyl 6- (N-methylanilino) hexanoate. bp: 141 to 143 ° C./0.9 mmHg NMR (CDCl ₃ ) δ: 1.25 (3 H, t, J = 7.2 Hz),
1.37 (2H, m), 1.65 (4H, m), 2.30 (2H, t,
J = 7.4Hz), 2.92 (3H, s), 3.30 (2H, t, J
= 7.4Hz), 4.13 (2H, q, J = 7.2Hz), 6.67
(3H, m), 7.22 (2H, m).

Reference Example 4 tert-butyl 4-chlorobutyrate tert-butanol 18.5 g and dimethylaniline 18.2 g
1 of 4-chlorobutyryl chloride with stirring under ice-cooling.
After dropwise adding 4.1 g, the mixture was stirred in an oil bath (100 ° C.) for 2 hours. To the reaction mixture were added 200 ml of ether, 100 ml of water and 50 ml of 1N hydrochloric acid, and the mixture was shaken to further add 1N to the organic layer.
-Washed with hydrochloric acid, sodium hydrogen carbonate solution and brine in this order and dried over anhydrous magnesium sulfate. After distilling off the solvent,
Distillation under reduced pressure gave 13.2 g of tert-butyl 4-chlorobutyrate. bp: 94-95 ° C./22 mmHg NMR (CDCl ₃ ) δ: 1.45 (9 H, s), 2.05 (2 H,
quintet, J = 7Hz), 2.41 (2H, t, J = 7Hz), 3.
59 (2H, t, J = 7Hz).

Reference Example 5 4- (N-methylanilino) butyric acid tert-butyl ester 4-chlorobutyric acid tert-butyl ester 1 obtained in Reference Example 4
A mixture of 0.72 g and 7.72 g of N-methylaniline was added to 10
Stir for 21 hours in an oil bath at 0 ° C. 50 ml of saturated sodium hydrogen carbonate solution was added to the reaction solution, extracted with 300 ml of ether, and dried over anhydrous magnesium sulfate. After concentration, it was subjected to vacuum distillation to obtain 4.40 g of 4- (N-methylanilino) butyric acid.
tert-Butyl was obtained. bp: 140-147 ° C./1.5 mmHg NMR (CDCl ₃ ) δ: 1.45 (9H, s), 1.86 (2H,
quintet, J = 7Hz), 2.26 (2H, t, J = 7Hz), 2.
92 (3H, s), 3.35 (2H, t, J = 7Hz), 6.6 ~
6.8 (3H, m), 7.1-7.3 (2H, m).

Reference Example 6 N- (3-tert-butoxycarbonylpropyl) -N-methyl-p-phenylenediamine 8.00 g of tert-butyl 4- (N-methylanilino) butyrate obtained in Reference Example 5 was added to 80% acetic acid. It was dissolved in 30 ml, and under ice-cooling stirring, a solution of 2.44 g of sodium nitrite in 3 ml of water was added dropwise at 10 ° C or lower. After stirring at the same temperature for 30 minutes, acetic acid 3
0 ml was added and 19 g of zinc powder was added at 10 ° C or lower. After stirring for 30 minutes, the reaction solution was filtered, the filtrate was concentrated under reduced pressure, and the mixture was shaken with aqueous sodium hydrogen carbonate and ether,
The ether layer was dried over anhydrous magnesium sulfate. After concentration under reduced pressure, the residue was subjected to silica gel column chromatography and developed with hexane / ethyl acetate = 2: 1 to give 3.21 g of N-tert-butoxycarbonylpropyl-N-methyl-p-phenylenediamine. Obtained as a brown oil. NMR (CDCl ₃ ) δ: 1.44 (9H, s), 1.81 (2H,
quintet, J = 7Hz), 2.55 (2H, t, J = 7Hz), 2.
50-3.50 (2H, br.), 2.80 (3H, s), 3.19
(2H, t, J = 7Hz), 6.65 (4H, s).

Reference Example 7 3- [N- (4-ethoxycarbonylbutyl) -N-methylamino] -7-dimethylaminophenazathionium
Chloride 1-N-hydrochloric acid 1 ml and water 14 ml were added to 0.25 g of ethyl 5- (N-methylanilino) valerate obtained in Reference Example 2 under ice-cooling and dissolved, and then 2-amino-5-dimethylaminophenylthiosulfone. Acid [Ann. Chem. , 251 , 1
(1888)] 0.25 g was added, and then a solution of 0.2 g of sodium dichromate dihydrate in 2 ml of water was added dropwise at 5 to 10 ° C., and the mixture was stirred at the same temperature for 30 minutes. Acetic acid 0.0 in the reaction solution
After adding 3 ml and stirring at room temperature for 1 hour, the precipitated precipitate was collected by filtration and washed with water. Next, the precipitate was suspended in 20 ml of acetonitrile, 0.25 g of manganese dioxide and a catalytic amount of copper sulfate were added, and the mixture was heated under reflux for 30 minutes. The reaction solution was filtered, and the filtrate was concentrated to dryness under reduced pressure. The residue was subjected to silica gel column chromatography, developed with a mixed solvent of acetonitrile and CH ₃ CN / H ₂ O / 1N HCl = 40: 5: 1, and then 3- [N- (4-ethoxycarbonylbutyl) was added.
0.29 g of -N-methylamino] -7-dimethylaminophenazathionium chloride was obtained. NMR (D ₂ O) δ: 1.28 (3H, t), 1.67 (4H,
m), 2.48 (2H, m), 3.17 (9H, br.s), 3.55
(2H, m), 4.20 (2H, q), 6.80 to 7.50 (6H,
m). IR (KBr) ν: 3420 (br.), 1725, 1600,
1490, 1440, 1390, 1340, 1245,
1180, 1140, 885 cm ^-1 .

Reference Example 8 3- [N- (4-ethoxycarbonylbutyl) -N-methylamino] -7-dimethylaminophenazathionium
Chloride 0.2 g of ethyl 5- (N-methylanilino) valerate obtained in Reference Example 2 was dissolved in 10 ml of 50% acetonitrile-water and 0.1 ml of acetic acid and 0.1 ml of 2-amino-5-dimethylaminophenylthiosulfonic acid. 2 g and manganese dioxide 0.5 g were added, and the mixture was stirred at room temperature for 2 hours. After stirring at 80 ° C for 5 hours, the reaction solution is filtered and washed with acetonitrile,
The filtrate was concentrated to dryness under reduced pressure. The residue was subjected to silica gel column chromatography, CH ₃ CN and then CH ₃ CN.
/ H ₂ O / 1N HCl = 40: 5: 1, 0.1
1 g of 3- [N- (4-ethoxycarbonylbutyl) -N-
Methylamino] -7-dimethylaminophenazathionium chloride was obtained.

Reference Example 9 3- [N- (4-carboxybutyl) -N-methylamino]
-7-Dimethylaminophenazathionium chloride 3- [N- (4-ethoxycarbonylbutyl) -N-methylamino] -7-dimethylaminophenazathionium
0.7 g of chloride was dissolved in 10 ml of 1N hydrochloric acid and reacted at room temperature for 48 hours. The reaction mixture was subjected to silica gel column chromatography, water, CH ₃ CN / H ₂ O = 10: 1.
Then, the mixture was developed in the order of CH ₃ CN / H ₂ O / 1N HCl = 40: 5: 1, and the target fraction was concentrated and lyophilized to give 3-
[N- (4-carboxybutyl) -N-methylamino] -7
-0. Of dimethylaminophenazathionium chloride.
7 g were obtained. NMR (D ₂ O) δ: 1.67 (4H, br.s), 2.49 (2H,
br.s), 3.08 (9H, br.s), 3.45 (2H, br.s), 6.
60-7.40 (6H, m). IR (KBr) ν: 3650 to 2800, 1720, 16
00, 1485, 1390, 1335, 885 cm ^-1 .

Example 1 3- [N-Methyl-N- (4-succinimidooxycarbonylbutyl) amino] -7-dimethylaminophenazathionium chloride 3- [N- (4-carboxy) obtained in Reference Example 9 Butyl) -N
0.34 g of -methylamino] -7-dimethylaminophenazathionium chloride was added to dimethylformamide 1
After dissolving in 00 ml and concentrating under reduced pressure to about 30 ml,
0.255 g of N-hydroxysuccinimide and 1-
Ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (0.425 g) was added, and the mixture was reacted at room temperature for 3 days. After evaporating the solvent under reduced pressure, the residue was dissolved in dichloromethane, washed with saturated brine, and dried over anhydrous sodium sulfate. After concentrating under reduced pressure, 6 ml of dichloromethane was added to the residue.
Was added and dissolved, and then 100 ml of ethyl acetate was added.
The deposited precipitate was collected by filtration and dried to give 3- [N-methyl-N-
(4-succinimidooxycarbonylbutyl) amino]
0.2 g of -7-dimethylaminophenazathionium chloride was obtained. NMR (DMSO-d ₆ ) δ: 1.74 (4H, br.s), 2.7
7 (2H, br.s), 2.82 (4H, s), 3.33 (3H, s),
3.38 (6H, s), 3.77 (2H, br.s), 7.45-7.
60 (4H, m), 7.80-7.98 (2H, m). IR (KBr) ν: 3425 (br.), 1810, 1780,
1735, 1600, 1395, 1355 (shoulder),
1340, 855 cm ^-1 .

Reference Example 10 3- [N- (5-Ethoxycarbonylpentyl) -N-methylamino] -7-dimethylaminophenazathionium chloride Ethyl 6- (N-methylanilino) hexanoate obtained in Reference Example 3 Under ice-cooling, 1 g of 1 N hydrochloric acid (4 ml) and water (50 ml) were added to the solution to dissolve it, then 2-amino-5-dimethylaminophenylthiosulfonic acid (1 g) was added, and then sodium dichromate dihydrate (0.8 g) in water (6 ml) was added. The solution was added dropwise at 5 to 10 ° C, and the mixture was stirred at the same temperature for 30 minutes and further at room temperature for 1 hour.
The precipitate was collected by filtration, washed with water, suspended in 80 ml of acetonitrile, added with 1 g of manganese dioxide and a catalytic amount of copper sulfate,
The mixture was heated under reflux for 1 hour. The reaction solution was filtered, the residue was washed with methanol, and then the filtrate was concentrated to dryness under reduced pressure. The residue was subjected to silica gel column chromatography, CH ₃ C
N was developed with CH ₃ CN / H ₂ O / 1N HCl = 40: 5: 1, and the desired fraction was lyophilized to give 3- [N- (5-ethoxycarbonylpentyl) -N-methylamino] -7- Dimethylaminophenazathionium chloride 0.87
5 g were obtained. NMR (CDCl ₃ ) δ: 1.26 (3H, t, J = 7Hz), 1.
49 (2H, m), 1.74 (4H, m), 2.35 (2H, t, J =
7.2Hz), 3.46 (6H, s), 3.71 (2H, br.t),
4.13 (2H, q, J = 7Hz), 7.28 (2H, m), 7.6
0-8.00 (4H, m). IR (KBr) ν: 3425 (br.), 1725, 1600,
1490, 1390, 1335, 1245, 1175,
1140, 885 cm ^-1 .

Example 2 3- [N-Methyl-N- (5-succinimidooxycarbonylpentyl) amino] -7-dimethylaminophenazathionium chloride 3- [N- (5- 0.785 g of ethoxycarbonylpentyl) -N-methylamino] -7-dimethylaminophenazathionium chloride was added to 1N-
It was dissolved in 10 ml of hydrochloric acid and reacted at room temperature for 16 hours. The reaction solution was subjected to silica gel column chromatography, water,
The mixture was developed in the order of CH ₃ CN / H ₂ O / 1N HCl = 40: 5: 1, and the target fraction was concentrated to dryness. The residue was dissolved in 200 ml of dimethylformamide, the insoluble material was filtered off, and then about 50 ml.
Concentrated to. To this solution, 0.54 g of N-hydroxysuccinimide and 0.9 g of WSC were added and reacted at room temperature for 48 hours. The reaction mixture was concentrated under reduced pressure, the residue was dissolved in dichloromethane, washed with saturated brine, and dried over anhydrous sodium sulfate. After concentrating under reduced pressure, 15 ml of residue
Was dissolved in dichloromethane, 250 ml of ethyl acetate was added, and the deposited precipitate was collected by filtration and dried to give 3- [N-methyl-N- (5-succinimidooxycarbonylpentyl).
0.718 g of amino] -7-dimethylaminophenazathionium chloride was obtained. NMR (CDCl ₃ ) δ: 1.56 (2H, m), 1.83 (4H,
m), 2.66 (2H, t, J = 6.8Hz), 2.91 (4H,
s), 3.40 (3H, s), 3.46 (6H, s), 3.73 (2)
H, m), 7.20-8.00 (6H, m). IR (KBr) ν: 3425, 1810, 1780, 17
35, 1600, 1390, 1335, 1210, 11
40, 1070, 885 cm ^-1 .

Reference Example 11 3- [N- (3-tert-butoxycarbonylpropyl)-
N-Methylamino] -7-dimethylaminophenazathionium chloride 0.50 g of tert-butyl 4- (N-methylanilino) butyrate obtained in Reference Example 5 was added to 0.41 g of potassium hydrogen sulfate under ice cooling.
Was dissolved in 25 ml of water, and 0.50 g of 2-amino-5-dimethylaminophenylthiosulfonic acid was added.
0.40 g of sodium dichromate dihydrate in this suspension solution
A 5 ml solution of water was added dropwise at 5-10 ° C and then acetic acid was added to 0.0
After adding 6 ml and stirring at the same temperature for 30 minutes, the mixture was further reacted at room temperature for 1.5 hours. The reaction solution was filtered and washed with water, then the residue was suspended in acetone, 4.0 g of manganese dioxide and 0.03 g of copper sulfate were added, and the mixture was heated under reflux for 1 hour. The reaction solution was filtered and then concentrated to dryness to give a crude product, which was subjected to silica gel column chromatography, and acetonitrile, 90% acetonitrile-water, and then acetonitrile /
When developed with water / 1N HCl = 30: 5: 3, 0.33 g of 3- [N- (3-tert-butoxycarbonylpropyl)-
N-methylamino] -7-dimethylaminophenazathionium chloride was obtained. mp: 94-98 ° C (dec.) NMR (D ₂ O) δ: 1.52 (9H, s), 1.87 (2H,
m), 2.43 (2H, t, J = 7Hz), 3.13 (3H, s),
3.17 (6H, s), 3.47 (2H, m), 6.81 (2H, br.
s), 7.0-7.1 (2H, m), 7.25-7.4 (2H, m). IR (KBr) ν: 3380, 1720, 1600, 13
90, 1335, 1140, 885, 615 cm ^-1 . λmax: 651 nm (in MeOH) Elemental analysis: Calculated as C ₂₃ H ₃₀ N ₃ O ₂ SCl · 2H ₂ O: C, 57.07; H, 7.08; N 8.68 Found: C, 57.08 H, 6.70; N8.70

Reference Example 12 3- [N- (3-carboxypropyl) -N-methylamino] -7-dimethylaminophenazathionium chloride 3- [N- (3-tert-butoxy) obtained in Reference Example 11 Carbonylpropyl) -N-methylamino] -7-dimethylaminophenazathionium chloride (0.05 g) was dissolved in trifluoroacetic acid (3 ml) and reacted at room temperature for 1 hour. After distilling off trifluoroacetic acid under reduced pressure, the residue was subjected to silica gel column chromatography and developed with acetonitrile and then with acetonitrile / water / 1N hydrochloric acid = 30: 5: 3,
0.014 g of 3- [N- (3-carboxypropyl) -N
-Methylamino] -7-dimethylaminophenazathionium chloride was obtained. NMR (D ₂ O) δ: 1.79 (2H, m), 2.51 (2H, m
m), 3.00 (9H, br.s), 3.31 (2H, m), 6.55
7.10 (6H, m). IR (KBr) ν: 3420, 1605, 1395, 13
40, 1140, 885, 850, 800 cm ^-1 .

Example 3 3- [N-Methyl-N- (3-succinimidooxycarbonylpropyl) amino] -7-dimethylaminophenazathionium chloride 3- [N- (3-tert -Butoxycarbonylpropyl) -N-methylamino] -7-dimethylaminophenazathionium chloride (0.17 g) was mixed with 15 ml of trifluoroacetic acid and reacted at room temperature for 15 minutes. Trifluoroacetic acid was distilled off under reduced pressure and the residue was dissolved in 10 ml of acetonitrile. To this, 0.19 ml of pyridine and 0.32 g of DSO were added, and the mixture was stirred at room temperature for 1.5 hours.
The reaction mixture was concentrated to dryness, the residue was dissolved in 50 ml of dichloromethane, shaken with saturated saline containing 5 ml of 1N hydrochloric acid, washed with saturated saline and dried over anhydrous sodium sulfate. After concentration under reduced pressure, the residue was dissolved in dichloromethane, an excess amount of ethyl acetate was added, and the resulting precipitate was collected by filtration and dried to give 0.12 g of 3- [N-methyl-
N- (3-succinimidooxycarbonylpropyl) amino] -7-dimethylaminophenazathionium chloride was obtained. mp: 125-130 ° C (dec.) NMR (CDCl ₃ ) δ: 2.11 (2H, m), 2.81 (2H,
t, J = 6Hz), 2.91 (4H, s), 3.37 (9H, br.
s), 3.79 (2H, m), 7.23 (2H, d, J = 9Hz),
7.75 (2H, d, J = 9Hz), 7.76 (2H, m). IR (KBr) ν: 3420, 1810, 1790, 17
35, 1600, 1390, 1340, 1140, 88
5 cm ^-1 .

Reference Example 13 2-amino-5- [N- (3-ethoxycarbonylpropyl) -N-methylamino] phenylthiosulfonic acid N- (3-ethoxycarbonylpropyl) obtained in Reference Example 1
To 8.97 g of -N-methyl-p-phenylenediamine was added 1N sulfuric acid (76 ml) under ice-cooling, and the mixture was dissolved. A solution of 88 g of water in 19 ml of water was added at 10 ° C. or lower, and then potassium dichromate of 3.65 g of water 57 was added.
The ml solution was added dropwise at 10 ° C. The cold bath was removed, the mixture was stirred at room temperature for 1 hour, and sodium hydrogen carbonate was added to adjust the pH to pH5. The reaction solution is filtered, the filtrate is concentrated to dryness under reduced pressure,
The residue was subjected to ODS (silanol octadecyl group silica gel as obtained by chemically binding) column chromatography and developed with 50% MeOH-H ₂ O, and lyophilized fractions purposes of 4g 2-Amino - 5- [N- (3-Ethoxycarbonylpropyl) -N-methylamino] phenylthiosulfonic acid was obtained. NMR (CH ₃ OD) δ: 1.22 (3H, t, J = 7Hz),
1.80 (2H, quintet, J = 7Hz), 2.35 (2H, t, J
= 7Hz), 2.96 (3H, s), 3.30 (2H, t, J = 7H)
z), 4.10 (2H, q, J = 7Hz), 6.96 (2H, br.
s), 7.23 (1H, br.s). IR (KBr) ν: 1730, 1625, 1505, 12
20, 1030, 835 cm ^-1 .

Reference Example 14 [N- (3-tert-butoxycarbonylpropyl) -N-
Methylamino]-[N ′-(3-ethoxycarbonylpropyl) -N′-methylamino] indaminethiosulfonic acid 0.997 g of tert-butyl 4- (N-methylanilino) butyrate obtained in Reference Example 5 was iced. Cold, potassium hydrogen sulfate 0.81
2-amino-5- [N- (3-ethoxycarbonylpropyl) -N-methylamino] phenylthiosulfonic acid obtained in Reference Example 13 was dissolved by adding a solution of 7 g of water in 10 ml.
394 g was added. Then 1.18 of potassium dichromate
g was added little by little and stirred at 5 to 10 ° C for 1 hour. The precipitate was collected by filtration, washed with water, dissolved in methanol, the insoluble material was filtered off, and the filtrate was concentrated under reduced pressure. Expand the concentrate with _{50% CH 3 CN-H 2} O and subjected to ODS column chromatography, the 0.6g [N- (3-tert- butoxycarbonyl-propyl) -N- methylamino] - [N' (3-Ethoxycarbonylpropyl) -N'-methylamino] indaminethiosulfonic acid was obtained. IR (KBr) ν: 3450, 3350, 1730, 16
10, 1515, 1365, 1240, 1170, 10
15, 820, 615 cm ^-1 .

Reference Example 15 3- [N- (3-tert-butoxycarbonylpropyl)-
N-Methylamino] -7- [N- (3-ethoxycarbonylpropyl) -N-methylamino] phenazathionium
Chloride of [N- (3-tert-butoxycarbonylpropyl) -N-methylamino]-[N ′-(3-ethoxycarbonylpropyl) -N′-methylamino] indaminethiosulfonic acid obtained in Reference Example 14 Dissolve 0.60 g in 25 ml of acetone,
3 g of manganese dioxide was added, and the mixture was stirred at room temperature for 20 hours.
The manganese dioxide was filtered off and washed with methanol. The filtrate was concentrated under reduced pressure, the residue was subjected to silica gel column chromatography, developed with acetonitrile / water = 20: 1, then with acetonitrile / water / 1N hydrochloric acid = 120: 20: 3, and the desired fractions were collected and concentrated. , 3- [N- (3-tert
-Butoxycarbonylpropyl) -N-methylamino]-
0.26 of 7- [N- (3-ethoxycarbonylpropyl) -N-methylamino] phenazathionium chloride
g was obtained. NMR (D ₂ O) δ: 1.30 (3H, t, J = 7Hz), 1.5
0 (9H, s), 1.83 to 1.90 (2H, br.), 2.38 to
2.55 (2H, m), 3.15 (6H, br.s), 3.47 (2H, m)
br.s), 4.20 (2H, q, J = 7Hz), 6.85 (2H, br.
s), 7.11 (2H, m), 7.65 (2H, m). IR (KBr) ν: 1725, 1600, 1400, 13
65, 1340, 1185, 1150, 890 cm ^-1 .

Example 4 3- [N-Methyl-N- (3-succinimidooxycarbonylpropyl) amino] -7- [N- (3-ethoxycarbonylpropyl) -N-methylamino] phenazathionium chloride Reference 3- [N- (3-tert-butoxycarbonylpropyl) -N-methylamino] -7- [N- (3-ethoxycarbonylpropyl) -N-methylamino] phenazathionium chloride obtained in Example 15 0 18 ml of trifluoroacetic acid was added to .24 g, and the mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated to dryness under reduced pressure, acetonitrile (15 ml), pyridine (0.33 ml) and DSC (0.52 g) were added to the residue, and the mixture was stirred at room temperature for 2 hr. The reaction solution was concentrated under reduced pressure, the residue was dissolved in 150 ml of dichloromethane, washed with a saturated saline solution containing 6 ml of 1N hydrochloric acid, further washed with a saturated saline solution, and dried with anhydrous sodium sulfate. The residue obtained by concentration to dryness was reprecipitated from dichloromethane-ethyl acetate to give 0.18 g.
Thus, 3- [N-methyl-N- (3-succinimidooxycarbonylpropyl) amino] -7- [N- (3-ethoxycarbonylpropyl) -N-methylamino] phenazathionium chloride was obtained. mp: 70-85 ° C. (dec.) NMR (CDCl ₃ ) δ: 1.27 (3 H, t, J = 7 Hz), 2.
04 (2H, m), 2.18 (2H, m), 2.53 (2H, t, J =
6.5 Hz), 2.89 (2 H, m), 2.97 (4 H, s), 3.
40 (3H, s), 3.42 (3H, s), 3.83 (4H, m),
4.16 (2H, q, J = 7Hz), 7.36 (2H, t, J = 10)
Hz), 7.59 (2H, d, J = 15Hz), 7.85 (2H, d
d, J = 2,10 Hz). IR (KBr) ν: 3420, 1810, 1780, 17
35, 1595, 1390, 1335, 1190, 11
40, 885 cm ^-1 .

Reference Example 16 2-Amino-5- [N- (3-tert-butoxycarbonylpropyl) -N-methylamino] phenylthiosulfonic acid N- (3-tert-butoxycarbonylpropyl) obtained in Reference Example 6. ) -N-Methyl-p-phenylenediamine 3.30 g
Was dissolved in 50 ml of 1N sulfuric acid under ice cooling, and while stirring, aluminum sulfate 14-18 hydrate 8.33 g of water 15 ml
Solution, sodium thiosulfate pentahydrate 9.31 g water 15 ml
A solution and then a solution of 1.27 g of potassium dichromate dihydrate in 17 ml of water were added successively at a reaction temperature of 5-10 ° C. After stirring at the same temperature for 30 minutes, the mixture was further stirred at room temperature for 1 hour. Sodium hydrogencarbonate was added to the reaction solution to adjust the pH to 4 and the mixture was concentrated to dryness under reduced pressure. 500 ml of methanol was added to the concentrate, the insoluble matter was filtered off, and the filtrate was concentrated under reduced pressure.
The residue was subjected to silica gel column chromatography, developed with ethyl acetate / methanol = 15: 1, then (5: 1), and 1.54 g of 2-amino-5- [N- (3-tert-butoxycarbonylpropyl). ) -N-Methylamino] phenylthiosulfonic acid was obtained as a dark green viscous material. NMR (CDCl ₃ ) δ: 1.42 (9H, s), 1.73 (2H,
m), 2.19 (2H, br.s), 2.76 (3H, br.s), 3.1
5 (2H, br.m), 4.50 (3H, br.s), 6.50-7.4
0 (3H, m).

Reference Example 17 Bis [N- (3-tert-butoxycarbonylpropyl)-
N-Methylamino] indaminethiosulfonic acid 1.16 g of tert-butyl 4- (N-methylanilino) butyrate obtained in Reference Example 5 was dissolved in 90 ml of a solution of 0.95 g of potassium hydrogensulfate in water and the mixture was cooled with ice for reference. 2-amino-5 obtained in Example 16
-[N- (3-tert-butoxycarbonylpropyl) -N
-Methylamino] phenylthiosulfonic acid 1.62 g was added, followed by potassium dichromate dihydrate 1.38 g water 1
The 0 ml solution was added dropwise at 5-10 ° C. After adding 0.14 ml of acetic acid and stirring for 1 hour at room temperature, the precipitate was collected by filtration and washed with water. The filter residue is extracted with 200 ml of methanol and then concentrated to dryness. The residue was subjected to silica gel column chromatography and developed with chloroform / methanol = 20: 1 to give 0.48 g of bis [N- (3-tert-butoxycarbonylpropyl) -N-methylamino] indaminethiosulfonic acid. Was obtained. NMR (CDCl ₃ ) δ: 1.45 (18H, s), 1.88 (4
H, br.), 2.86 (4H, br.s), 2.90-3.20 (4
H, m), 3.20 to 3.70 (6H, br.), 6.50 to 7.7
0 (7H, m).

Reference Example 18 3,7-Bis [N- (3-tert-butoxycarbonylpropyl) -N-methylamino] phenazathionium chloride Bis [N- (3-tert-butoxy) obtained in Reference Example 17 0.47 g of (carbonylpropyl) -N-methylamino] -indaminethiosulfonic acid was dissolved in 40 ml of acetone, 2.0 g of manganese dioxide and 0.03 g of copper sulfate were added, and the mixture was heated under reflux for 30 minutes. The reaction solution was filtered, and the filtrate was concentrated to dryness under reduced pressure. The residue was subjected to silica gel column chromatography, acetonitrile, 90% acetonitrile-water, then acetonitrile / water / 1N HCl = 120: 20: 3.
The target fraction was concentrated to dryness. The residue was reprecipitated from dichloromethane-ethyl acetate to give 3,7-bis [N-
0.11 g of (3-tert-butoxycarbonylpropyl) -N-methylamino] phenazathionium chloride was obtained. mp: 142-145 ° C NMR (CDCl ₃ ) δ: 1.47 (18H, s), 2.00 (4
H, quintet, J = 7Hz), 2.43 (4H, t, J = 7Hz),
3.43 (6H, s), 3.79 (4H, t, J = 7Hz), 7.3
9 (2H, d, J = 10Hz), 7.62 (2H, br.s), 7.9
1 (2H, d, J = 10Hz). IR (KBr) ν: 3430, 2970, 2920, 17
20, 1595, 1395, 1335, 1240, 11
40,980 cm ^-1 . Elemental _{analysis: C 30 H 42 N 3 O} 4 SCl · 3H 2 O Calculated: C, 57.17; H, 7.68 ; N, 6.67 Found: C, 57.29; H, 7 . 45; N, 6.67.

Reference Example 19 3,7-Bis [N- (3-carboxypropyl) -N-methylamino] phenazathionium chloride 3,7-Bis [N- (3-tert- Butoxycarbonylpropyl) -N-methylamino] phenazathionium chloride (0.11 g) was dissolved in 10 ml of trifluoroacetic acid, reacted at room temperature for 30 minutes, and then concentrated to dryness under reduced pressure to give 3,7-bis (3-bis-bis (3-bis-bis (aminobenzophen))). [N- (3-carboxypropyl) -N-methylamino] phenazathionium chloride was obtained quantitatively as the trifluoroacetate salt. NMR (D ₂ O) δ: 1.86 (4H, br.s), 2.45 (4H, 4H, br.s)
br.s), 2.80 to 3.70 (10H, br.m), 6.70 to 7.
40 (6H, br.m).

Example 5 3,7-Bis [N-methyl-N- (3-succinimidooxycarbonylpropyl) amino] phenazathionium
Chloride and 3- [N-methyl-N- (3-succinimidooxycarbonylpropyl) amino] -7- [N
-(3-Carboxypropyl) -N-methylamino] phenazathionium chloride 3,7-bis [N- (3-carboxypropyl) -N-methylamino] phenazathionium chloride obtained in Reference Example 19 0.11 g of fluoroacetate was dissolved in 20 ml of acetonitrile, 0.14 ml of pyridine and DSC were added.
0.21 g was added, and the mixture was stirred at room temperature for 3 hours. After the reaction solution was concentrated under reduced pressure, dichloromethane was added to the residue to dissolve it,
The extract was washed with saturated saline and dried over anhydrous sodium sulfate. The deep blue powder obtained by concentration to dryness was subjected to Sephadex (registered trademark) LH-20 column chromatography, and when developed with acetonitrile, the bissuccinimide ester derivative 3,7-bis [N-methyl] was eluted first. -N-
0.1 g of (3-succinimidooxycarbonylpropyl) amino] phenazathionium chloride is then eluted, and then 3- [N] of the monosuccinimide ester derivative is eluted.
-Methyl-N- (3-succinimidooxycarbonylpropyl) amino] -7- [N- (3-carboxypropyl) -N-methylamino] phenazathionium chloride was 0.03 g, and each eluted fraction was concentrated. Obtained by drying.

[Bissuccinimide ester] mp: 115-120 ° C. (dec.) NMR (DMSO-d ₆ ) δ: 1.88 (2H, m), 2.02 (2
H, m), 2.38 (2H, t, J = 7Hz), 2.87 (4H,
s), 3.13 (2H, t, J = 7Hz), ca 3.30 (3H, s),
3.36 (3H, s), 3.76 (4H, m), 7.55 (4H, br.
s), 7.95 (2H, d, J = 10Hz). IR (KBr) ν: 3410, 2930, 1810, 17
75, 1735, 1595, 1390, 1335, 11
90, 1140, 880 cm ^-1 . [Monosuccinimide ester] mp: 129 to 133 ° C. (dec.) NMR (DMSO-d ₆ ) δ: 2.01 (4H, m), 2.84
(4H, s), 2.88 (4H, t, J = 7Hz), ca 3.3 (6H,
s), 3.80 (4H, m), 7.56 (4H, br.s), 7.96
(2H, d, J = 10Hz). IR (KBr) ν: 3400, 2930, 1810, 17
75, 1730, 1595, 1390, 1335, 11
90, 1140, 1065, 880 cm ^-1 .

Example 6 3- [N-methyl-N- (3-succinimidooxycarbonylpropyl) -amino] -7- [N- [3-
(1,3-Dicarboxypropylcarbamoyl) -propyl] -N-methylamino] phenazathionium chloride 3,7-bis [N-methyl-N- (3-succinimidooxycarbonylpropyl) obtained in Example 5 Amino] phenazathionium chloride (20 mg) was dissolved in 2 ml of acetonitrile, a solution of glutamic acid (8 mg), triethylamine (15 μl) and 50% acetonitrile-water (2 ml) was added, and the mixture was stirred at room temperature for 10 minutes. After adding 0.1 ml of 1N hydrochloric acid to the reaction solution, Sephadex (Sephadex) is added.
dex) (registered trademark) LH-20 column chromatography and developed with acetonitrile. The target fraction was concentrated to dryness, and 3- [N-methyl-N- (3-succinimidooxycarbonylpropyl) amino] -7- [N-
[3- (1,3-dicarboxypropylcarbamoyl)
10.8 mg of propyl] -N-methylamino] phenazathionium chloride was obtained as a blue powder. NMR (DMSO-d ₆ ) δ: 1.75 to 2.05 (6H,
m), 2.20 to 2.35 (4H, m), 2.84 (4
H, s), 2.85 to 2.95 (2H, m), 3.60 to
3.85 (6H, m), 4.15-4.3 (1H, m),
7.50 to 7.60 (3H, m), 7.9 to 8.1 (3H,
m). IR (KBr) ν: 3420, 2970, 2940, 267
0, 2490, 1810, 1735, 1660, 159
5, 1390, 1340, 1145, 1035, 880
cm ^-1 .

Reference Example 20 N- (5-ethoxycarbonylpentyl) -N-methyl-
p-Phenylenediamine N- (5-ethoxycarbonylpentyl) obtained in Reference Example 3
-N-methylaniline 10.0 g 80% acetic acid 41.3 ml
A solution of 3.8 ml of water containing 3.05 g of sodium nitrite was added at 10 ° C. or lower while stirring. Then, 37.5 ml of acetic acid was added to the reaction solution, and 23.8 g of zinc dust was added over 40 minutes while keeping the temperature below 10 ° C. The reaction solution was poured into 200 ml of ice water, filtered, and the zinc powder was washed with ethyl acetate. Ethyl acetate was added to the filtrate for extraction, the extract was neutralized with an aqueous sodium hydrogen carbonate solution, washed with saturated brine, and dried over anhydrous magnesium sulfate. After concentration under reduced pressure, the residue was subjected to silica gel column chromatography and developed with hexane / ethyl acetate = 2: 1 to obtain 2.76 g of the desired compound as a reddish purple viscous oil. NMR (CDCl ₃ ) δ (ppm): 1.25 (3H, t, J = 7.0H
z), 1.33 (2H, quintet, J = 7.5Hz), 1.54 (2
H, quintet, J = 7.5Hz), 1.65 (2H, quintet, J
= 7.5Hz), 2.29 (2H, t, J = 7.5Hz), 2.80
(3H, s), 2.90 to 3.50 (2H, br.), 3.16 (2
H, t, J = 7.5Hz), 4.12 (2H, q, J = 7.0Hz),
6.65 (5H, s). IR (neat) ν: 3430, 3350, 2930, 1730,
1515, 1260, 1175, 810 cm ^-1 .

Reference Example 21 3,7-Bis [N- (5-ethoxycarbonylpentyl)-
N-methylamino] phenazathionium chloride N- (5-ethoxycarbonylpentyl) -N-methyl-
1.32 g of p-phenylenediamine was dissolved in 65 ml of water containing 0.75 g of sulfuric acid, and while stirring under ice-cooling, a solution of 5 ml of water containing 2.18 g of sodium thiosulfate pentahydrate and 0.86 g of manganese dioxide. Was added. After 20 minutes, 4.36 g of sodium thiosulfate pentahydrate and 1.92 g of manganese dioxide were added. After stirring for 1 hour, add 1 reaction mixture.
The pH was adjusted to 4 with N-sulfuric acid, and N- (5-ethoxycarbonylpentyl) -N-methylaniline 1.70 g of 1N sulfuric acid 1
0 ml solution was added at the same temperature. Further, 2.08 g of manganese dioxide and 6 ml of 25% sulfuric acid were added, and the mixture was stirred for 1.5 hours. Then, the reaction solution was adjusted to pH with 1N sodium hydroxide.
Adjusted to 5, 0.77 g of manganese dioxide and 0.7 of copper sulfate.
29 g was added, and the mixture was stirred at 90 ° C. for 40 minutes. The reaction solution was filtered, the residue was washed with water, and then extracted with 300 ml of acetone. After the acetone extract was concentrated, the residue was extracted with chloroform and dried over anhydrous sodium sulfate. The residue after concentration was subjected to silica gel column chromatography, acetonitrile was added, and then 90% acetonitrile-
Water and acetonitrile / water / 1N hydrochloric acid = 120: 20: 3 were developed in this order to obtain 30 mg of the target compound. NMR (CDCl ₃ ) δ (ppm): 1.26 (6H, t, J = 7.0H
z), 1.50 (4H, m), 1.72 (4H, m), 2.35 (4)
H, t, J = 7.0Hz), 3.41 (6H, s), 3.71 (4H,
m), 4.13 (4H, q, J = 7.0Hz), 7.23 (2H, br.
d, J = 9.0 Hz), 7.80 (2H, br.s), 7.91 (2H,
br.d, J = 9.0 Hz). IR (KBr) ν: 1720, 1600, 1395, 1340,
1,140,885 cm ^-1

Reference Example 22 3,7-Bis [N- (5-carboxypentyl) -N-methylamino] phenazathionium chloride 3,7-bis [N- (5-ethoxycarbonylpentyl)-
N-methylamino] phenazathionium chloride
0.32 g was dissolved in 20 ml of 90% acetonitrile, 40 ml of 1N hydrochloric acid was added, and the mixture was stirred at room temperature for 20 hours.
The reaction mixture was concentrated under reduced pressure, and the residue was subjected to silica gel column chromatography, 90% acetonitrile-water,
Then, it was developed in the order of acetonitrile / water / 1N hydrochloric acid = 30: 5: 1 to obtain 0.32 g of the desired compound as a dark blue powder. NMR (DMSO-d ₆ ) δ (ppm): 1.25 to 1.50 (4H,
m), 1.50 to 1.75 (8H, m), 2.24 (4H, t, J =
7.0Hz), 3.50 (6H, s), 3.65-3.80 (4H,
m), 7.35 to 7.50 (2H, m), 7.54 (2H, br.s),
7.93 (2H, br.d, J = 9.0Hz). IR (KBr) ν: 1635, 1600, 1485, 1390,
1340,1240,1145,885 cm ^-1 .

Example 7 3,7-Bis [N-methyl-N- (5-succinimidooxycarbonylpentyl) amino] phenazathionium
Chloride and 3- [N-methyl-N- (5-succinimidooxycarbonylpentyl) amino] -7- [N-
(5-Carboxypentyl) -N-methylamino] phenazathionium chloride Method A 3,7-bis [N- (5-carboxypentyl) -N-methylamino] phenazathionium chloride 0.15 g was trifluoro It was dissolved in 10 ml of acetic acid and then concentrated to dryness. The trifluoroacetic acid salt obtained was dissolved in 30 ml of acetonitrile, 0.92 g of DSC and 0.85 of pyridine.
ml was added, and the mixture was stirred at room temperature for 15 hours. The reaction solution was concentrated under reduced pressure, dichloromethane was added to dissolve the solution, and the solution was washed twice with saturated saline and then dried over anhydrous sodium sulfate.
After concentration, the residue was subjected to Sephadex (registered trademark) LH-20 column chromatography and developed with acetonitrile, and 38 mg of the bissuccinimide ester derivative was used as the first fraction, and further 36 mg of the monosuccinimide ester derivative was used. Was obtained as the second fraction.

Method B 0.37 g of 3,7-bis [N- (5-carboxypentyl) -N-methylamino] phenazathionium chloride was dissolved in 5 ml of dimethylformamide, and 0.42 g of N-hydroxysuccinimide was dissolved. And WSC (0.70 g) were added and the mixture was stirred at room temperature for 3 hours. The reaction solution was concentrated to dryness under reduced pressure and then extracted with dichloromethane. The dichloromethane layer was washed twice with saturated saline and dried over anhydrous sodium sulfate.
After concentration, the residue is Sephadex LH-2
It was subjected to 0 column chromatography, developed with acetonitrile, and the target fraction was concentrated. The concentrate was dissolved in a small amount of dichloromethane and ethyl acetate was added to obtain 83 mg of a bissuccinimide ester derivative as a dark blue-purple powder.

[Bissuccinimide ester] NMR (DMSO-d ₆ ) δ (ppm): 1.40 to 1.55 (4H,
m), 1.65 to 1.75 (8H, m), 2.71 (4H, t, J =
7.0Hz), 2.82 (8H, s), 3.31 (6H, s), 3.7
5 (4H, br.t, J = 7.0Hz), 7.51 (2H, br.s),
7.54 (2H, br.d, J = 9.5Hz), 7.93 (2H, d, J
= 9.5 Hz). IR (KBr) ν: 1810, 1780, 1735, 1595,
1390, 1335, 1200, 1135, 1065, 88
0 cm ^-1 . [Monosuccinimide ester] NMR (DMSO-d ₆ ) δ (ppm): 1.35 to 1.65 (4H,
m), 1.65 to 1.80 (8H, m), 2.24 (2H, t, J =
7.0Hz), 2.71 (2H, t, J = 7.0Hz), 2.82
(4H, s), 3.35 (6H, s), 3.74 (4H, br.t, J =
7.0Hz), 7.50 (2H, s), 7.52 (2H, br.d, J =
9.5Hz), 7.92 (2H, d, J = 9.5Hz), IR (KBr) ν: 1810,1780,1735,1595,
1390, 1335, 1200, 1135, 1065, 88
0 cm ^-1 .

Example 8 3- [N-Methyl-N- (5-succinimidooxycarbonylpentyl) amino] -7- [N- [5- (1,3-dicarboxypropylcarbamoyl) pentyl] -N-methyl Amino] phenazathionium chloride 3,7-bis [N-methyl-N- (5-succinimidooxycarbonylpentyl) amino] phenazathionium
To a solution of 72 mg of chloride in 6 ml of acetonitrile was added a solution prepared from 27 mg of glutamic acid, 3 ml of water and 51 μl of triethylamine. 5 minutes later, 1N hydrochloric acid 0.35 m
Sephadex (registered trademark) LH-
After 20 column chromatography and developing with acetonitrile, 16 mg of the desired compound was obtained. NMR (DMSO-d ₆ ) δ (ppm): 1.35 to 1.60 (4H,
m), 1.60 to 1.80 (8H, m), 1.80 to 2.00 (2
H, m), 2.15 (2H, t, J = 6.0Hz), 2.27 (2H,
t, J = 7.0Hz), 2.71 (2H, t, J = 7.0Hz), 2.
82 (4H, s), 3.35 (6H, s), 3.72 (4H, br.
s), 4.20 (1H, m), 7.40 to 7.55 (4H, m), 7.
92 (1H, d, J = 9.5Hz), 8.04 (1H, d, J = 9.
5 Hz). IR (KBr) ν: 1810, 1780, 1735, 1650,
1595, 1390, 1335, 1235, 1200, 11
35,1065,880 cm ^-1 .

Reference Example 23 N- (3-maleimidopropyl) -N-methylaniline N- (3-aminopropyl) -N-methylaniline [S. Shapiro et al., J. Am. .Chem.
Soc., 81 , 3081 (1958)] was dissolved in 50 ml of dichloromethane, 6 g of maleic anhydride was added little by little under ice cooling, and the mixture was stirred at room temperature for 1 hour. After the solvent was distilled off, 20 ml of acetic anhydride was added and the mixture was left for 2 days at room temperature. The reaction solution was stirred at 60 ° C. for 2 hours and further at 100 ° C. for 2 hours, concentrated under reduced pressure, and the residue was extracted with ethyl acetate. The organic layer was neutralized with an aqueous sodium hydrogen carbonate solution, washed with saturated brine, and dried over anhydrous sodium sulfate. The residue after concentration was subjected to silica gel column chromatography and developed with hexane / ethyl acetate = 4: 1. The obtained target compound is recrystallized from the same solvent,
4.1 g of yellow needle crystals were obtained. mp: 87-88 ° C NMR (CDCl ₃ ) δ (ppm): 1.89 (2H, quintet, J =
7.3 Hz), 2.91 (3 H, s), 3.33 (2 H, t, J = 7.
3Hz), 3.57 (2H, t, J = 7.3Hz), 6.68 (3
H, m), 6.69 (2H, s), 7.21 (2H, m). IR (KBr) ν: 1700, 1600, 1510, 1410,
1370, 1220, 830, 750, 690 cm ^-1 . Elemental _{analysis: C 14 H 16 N 2 O} 2 Calculated: C, 68.83; H, 6.60 ; N, 11.47 Found: C, 68.95; H, 6.77 : N, 11 .49.

Reference Example 24 3- [N- (3-maleimidopropyl) -N-methylamino] -7- [N- (3-ethoxycarbonylpropyl) -N
-Methylamino] phenazathionium chloride N- (3-maleimidopropyl) -N-methylaniline
0.25 g was dissolved in 10 ml of acetone and 1 ml of 1N hydrochloric acid and 9 ml of water were added. Then 2-amino-5- [N- (3-
Ethoxycarbonylpropyl) -N-methylamino] phenylthiosulfonic acid (0.35 g) was added, and sodium dichromate dihydrate (0.2 g) in water 2 was added while stirring under ice cooling.
The ml solution was added dropwise. After stirring at the same temperature for 30 minutes,
0.03 ml of acetic acid was added, and the mixture was further stirred at room temperature for 30 minutes. Next, a catalytic amount of copper sulfate and 0.25 g of manganese dioxide were added, and the mixture was stirred at room temperature for 30 minutes and further at 70 ° C. for 1 hour. After cooling, the reaction solution was filtered, the residue was washed with ethanol, and the filtrate was concentrated under reduced pressure. The residual portion was re-extracted with ethanol and the insoluble matter was filtered off. The filtrate was concentrated and then subjected to silica gel column chromatography and developed with acetonitrile and then with acetonitrile / water / 1N hydrochloric acid = 40: 5: 1 to obtain 0.31 g of the desired compound. NMR (CDCl ₃ ) δ (ppm): 1.28 (3H, t, J = 7.2H
z), 1.78 (4H, m), 2.55 (2H, m), 3.39 (3
H, s), 3.46 (3H, s), 3.60 to 3.95 (6H, m),
4.17 (2H, q, J = 7.2Hz), 6.80 (2H, s), 7.
15 to 8.00 (6H, m), IR (KBr) ν: 1720, 1700, 1590, 1390,
1330, 1225, 1180, 1140, 880 cm ^-1 .

Example 9 3- [N- (3-maleimidopropyl) -N-methylamino] -7- [N- (3-carboxypropyl) -N-methylamino] phenazathionium chloride 3- [N -(3-maleimidopropyl) -N-methylamino] -7- [N- (3-ethoxycarbonylpropyl) -N
-Methylamino] phenazathionium chloride 0.
3 g was dissolved in a mixture of 2 ml of acetonitrile and 6 ml of 1N hydrochloric acid and left open for 2 days. The deposited precipitate having a metallic luster was collected by filtration, washed with ether and dried to obtain 0.25 g of the desired compound. NMR (DMSO-d ₆ ) δ (ppm): 1.90 (4H, m), 2.3
9 (2H, t, J = 6.8Hz), 3.23 (3H, s), 3.35
(3H, s), 3.54 (2H, m), 7.01 (2H, s), 7.4
0 to 7.65 (4H, m), 7.85 to 8.00 (2H, m). IR (KBr) ν: 1735, 1700, 1595, 1390,
1330 cm ^-1 .

Example 10 3- [N- (3-maleimidopropyl) -N-methylamino] -7- [N- [3- (1,3-dicarboxypropylcarbamoyl) propyl] -N-methylamino] Phenazathionium chloride 3- [N- (3-maleimidopropyl) -N-methylamino] -7- [N- (3-carboxypropyl) -N-methylamino] phenazathionium chloride 51 mg 4Å
Was dissolved in 5 ml of dimethylformamide dried with a molecular sieve, and 25 mg of N-hydroxysuccinimide and 50 mg of WSC were added, and the mixture was reacted at room temperature for 23 hours. This solution was added to a solution prepared from 17.7 mg of L-glutamic acid, 5 ml of dimethylformamide, 0.5 ml of water and 34 μl of triethylamine under ice cooling and stirred for 1 hour. After concentrating under reduced pressure, the residue was dissolved in 5 ml of water and 0.24 ml of 1N hydrochloric acid was added. This solution was subjected to silica gel column chromatography and eluted with acetonitrile / water = 9: 1 and then with acetonitrile / water / 1N hydrochloric acid = 30: 15: 1. After the desired fraction was concentrated, the remaining portion was separated with Sephadex (registered trademark) L.
It was subjected to H-20 column chromatography, developed with acetonitrile / water = 10: 1, and the target fraction was lyophilized to obtain 26 mg of the target compound. NMR (D ₂ O) δ (ppm): 1.60 to 2.30 (6 H, m), 2.
45 (4H, m), 3.14 (6H, s), 3.35 to 3.65 (6
H, m), 4.35 (1H, t), 6.80 to 7.50 (6H, m),
6.92 (2H, s). IR (KBr) ν: 1730, 1700, 1640, 1595,
1390, 1330 cm ^-1 .

Reference Example 25 N- (3-Cyanopropyl) -N-methylaniline To 25 g of N-methylaniline was added 25 ml of 4-chlorobutyronitrile, and the mixture was stirred at 110 ° C. for 24 hours. After cooling, it was neutralized with an aqueous sodium hydrogen carbonate solution and extracted with ethyl acetate. The organic layer was washed with saturated saline and dried over anhydrous sodium sulfate. After concentration, the residue was distilled under reduced pressure to obtain 21.5 g of the desired compound as a pale yellow oily substance. bp: 118 to 121 ° C./0.5 mmHg NMR (CDCl ₃ ) δ (ppm): 1.96 (2H, quintet, J =
7.0Hz), 2.40 (2H, t, J = 7.0Hz), 2.96
(3H, s), 3.47 (2H, t, J = 7.0Hz), 6.74 (3
H, m), 7.25 (2H, m). IR (neat) ν: 2950, 2875, 2825, 2250,
1600, 1505, 1370, 1190, 750, 690c
m ^-1 .

Reference Example 26 N- (4-Aminobutyl) -N-methylaniline To 50 ml of dry ether was added 2 g of lithium aluminum hydride under a nitrogen stream, and N- (3-cyanopropyl) -N- was stirred while stirring. A solution of 9 g of methylaniline in 10 ml of dry ether was added dropwise. After stirring for 1 hour, the mixture was heated under reflux for 2 hours. After decomposing the excess reducing agent with ethyl acetate, the organic layer was washed with water and dried over anhydrous sodium sulfate. This was concentrated to obtain 9.8 g of the target compound as a colorless oily substance. NMR (CDCl ₃ ) δ (ppm): 1.54 (4H, m), 2.73
(2H, t, J = 7.0Hz), 2.93 (3H, s), 3.33 (2
H, t, J = 7.0 Hz), 6.70 (3 H, m), 7.22 (2 H,
m).

Reference Example 27 N- (4-maleimidobutyl) -N-methylaniline N- (4-aminobutyl) -N-methylaniline (7.2 g) was dissolved in 70 ml of dichloromethane, and 4 g of maleic anhydride was added at room temperature. Stir for 2 hours. After the solvent was distilled off, 10 ml of acetic anhydride was added, and the mixture was stirred at 100 ° C for 5 hours. After concentration, a sodium hydrogen carbonate aqueous solution was added to the residue to neutralize it, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure.
The residue was subjected to silica gel column chromatography,
When developed with hexane / ethyl acetate = 2: 1, 2.0 g of the target compound was obtained as yellow needle crystals. mp: 66-67 ° C NMR (CDCl ₃ ) δ (ppm): 1.60 (4H, m), 2.91
(3H, s), 3.33 (2H, t, J = 6.8Hz), 3.54 (2
H, t, J = 6.8Hz), 6.68 (2H, s), 6.68 (3H,
m), 7.22 (2H, m). IR (KBr) ν: 1695, 1600, 1505, 1410,
1365,750,690 cm ^-1 . Elemental _{analysis: C 15 H 18 N 2 O} 2 Calculated: C, 69.74; H, 7.02 ; N, 10.84 Found: C, 69.98; H, 7.20 ; N, 10 .92.

Reference Example 28 N- (4-ethoxycarbonylbutyl) -N-methyl-p
-Phenylenediamine N- (4-ethoxycarbonylbutyl) -obtained in Reference Example 2-
2.35 g of N-methylaniline was dissolved in 30 ml of 1N hydrochloric acid, and a 5 ml solution of water containing 0.76 g of sodium nitrite was added dropwise at 0 ° -4 ° C. After stirring at the same temperature for 30 minutes, 3
N hydrochloric acid (15 ml) and then zinc dust (2 g) were added at 20 to 27 ° C., and the mixture was further stirred at the same temperature for 1 hr. Sodium hydrogen carbonate was added to the reaction solution to neutralize it, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The concentrate was subjected to silica gel flash chromatography and developed with hexane / ethyl acetate = 1: 1 to obtain 1.7 g of the desired compound as a brown oily substance. NMR (CDCl ₃ ) δ (ppm): 1.25 (3H, t, J = 7.2H
z), 1.61 (4H, m), 2.31 (2H, t, J = 7.1H
z), 2.81 (3H, s), 3.18 (2H, t, J = 6.5H
z), 4.12 (2H, q, J = 7.2Hz), 6.65 (4H,
s).

Reference Example 29 2-Amino-5- [N- (4-ethoxycarbonylbutyl) -N-methylamino] phenylthiosulfonic acid N- (4-ethoxycarbonylbutyl) -N-methyl-p
1.7 g of phenylenediamine was dissolved in 13.6 ml of 1N hydrochloric acid, 10 ml of water containing 2 g of aluminum sulfate 14-18 hydrate was added, and the mixture was ice-cooled. To this, add 8 ml of water containing 2 g of sodium thiosulfate pentahydrate, and then add 5 ml of water containing 0.798 g of sodium dichromate dihydrate.
The solution was added dropwise. After stirring for 30 minutes under ice-cooling, 0.6 ml of acetic acid was added, and the mixture was further stirred at room temperature for 2 hours. After the reaction solution was concentrated to dryness, it was made alkaline with an aqueous solution of sodium hydrogen carbonate, and the insoluble material was filtered off. The filtrate is acetic acid at pH 4
After adjusting to, the mixture was concentrated to about 50 ml and cooled. The precipitated crystalline precipitate was collected by filtration, washed with cold water and dried to obtain 2.1 g of the desired compound. NMR (DMSO-d ₆ + D ₂ O) δ: 1.64 (3H, t, J =
7.0Hz), 1.51 (4H, m), 2.30 (2H, m), 3.0
1 (3H, s), 3.39 (2H, m), 4.04 (2H, q, J =
7.0 Hz), 6.80 to 7.40 (3 Hz, 3). IR (KBr) ν: 1720, 1620, 1250, 1180,
1110,630 cm ^-1 .

Reference Example 30 3- [N- (4-maleimidobutyl) -N-methylamino]
-7- [N- (4-Ethoxycarbonylbutyl) -N-methylamino] phenazathionium chloride 0.26 g of N- (4-maleimidobutyl) -N-methylaniline obtained in Reference Example 27 was added to 10 ml of acetone. Dissolve
1N hydrochloric acid (1 ml) and water (9 ml) were added and the mixture was ice-cooled. 2 to this
-Amino-5- [N- (4-ethoxycarbonylbutyl) -N-methylamino] phenylthiosulfonic acid 0.4
Then, a solution of 1 g of water containing 0.2 g of sodium dichromate dihydrate was added, and the mixture was stirred for 30 minutes. Acetic acid 0.03 ml
Was added, and the mixture was further stirred at room temperature for 1 hour. Then, catalytic amounts of 0.25 g of copper sulfate and manganese dioxide were added to the reaction solution, and the mixture was stirred at room temperature for 30 minutes and further at 70 ° C. for 1 hour. After cooling, the reaction solution was filtered, and the residue was washed with methanol and water. The filtrate was concentrated to dryness, shaken with saturated saline and chloroform, and the organic layer was dried over anhydrous sodium sulfate. After concentration, it was subjected to silica gel column chromatography, and then acetonitrile, then acetonitrile / water /
The mixture was developed in the order of 1N hydrochloric acid = 40: 5: 1, and the target fraction was concentrated. Acetonitrile was added to the concentrate, the insoluble matter was filtered off, and the filtrate was concentrated to dryness to give 0.289 g of the desired compound.
was gotten. NMR (CDCl ₃ ) δ (ppm): 1.26 (3H, t, J = 7.0H
z), 1.77 (8H, m), 2.41 (2H, t, J = 5.7H
z), 3.40 (3H, s), 3.61 (2H, t, J = 6.0H
z), 3.77 (4H, m), 4.13 (2H, q, J = 7.0H
z), 6.78 (2H, s), 7.65 to 7.96 (6H, m). IR (KBr) ν: 1720, 1700, 1595, 1390,
1330,1140,880 cm ^-1 .

Example 11 3- [N- (4-maleimidobutyl-N-methylamino]]
-7- [N- (4-Carboxybutyl) -N-methylamino] phenazathionium chloride 3- [N- (4-maleimidobutyl) -N-methylamino]
-7- [N- (4-Ethoxycarbonylbutyl) -N-methylamino] phenazathionium chloride 0.287
To g, 2 ml of acetonitrile and 6 ml of 1N hydrochloric acid were added and dissolved, and the mixture was left standing for 44 hours. A precipitate having a metallic luster is collected by filtration, washed with ether, and dried to give 0.24.
6 g of the expected compound are obtained. NMR (DMSO-d ₆ ) δ (ppm): 1.61 (8H, m), 2.3
0 (2H, t, J = 6.0Hz), 3.76 (4H, m), 7.03
(2H, s), 7.52 (4H, br.s), 7.85-8.00 (2
H, m). IR (KBr) ν: 1740, 1700, 1390, 1330c
m ^-1 .

Example 12 3- [N- (4-maleimidobutyl) -N-methylamino]
-7-Dimethylaminophenazathionium chloride 0.26 g of N- (4-maleimidobutyl) -N-methylaniline obtained in Reference Example 27 was dissolved in 10 ml of acetone to give 1N.
1 ml of hydrochloric acid and 10 ml of water were added, and the mixture was ice-cooled. 2-amino-5-dimethylaminophenyl thiosulfonic acid 0.25g
Sodium dichromate dihydrate 0.2g
2 ml of water containing water was added dropwise. After stirring for 30 minutes at the same temperature, 0.03 ml of acetic acid was added, and the mixture was stirred for another hour at room temperature. Acetone was distilled off under reduced pressure, and the precipitated insoluble material having metallic luster was collected by filtration and washed with water. This was suspended in 30 ml of 50% acetone-water, catalytic amounts of copper sulfate and manganese dioxide (025 g) were added, and the mixture was further heated at room temperature for 1 hour and further 70
Stir at ℃ for 1 hour. After cooling, the reaction solution was filtered and the residue was washed with acetone, water, and ethanol in this order. The filtrate is concentrated to dryness and then subjected to silica gel column chromatography, acetonitrile, then acetonitrile / water / 1N
Hydrochloric acid was developed in the order of 40: 5: 1, and the target fraction was concentrated. 50 ml of chloroform and 4 ml of ethanol in the residue
Was added, the insoluble matter was filtered off, and the filtrate was concentrated to dryness to give 0.4
2 g of the expected compound are obtained. NMR (DMSO-d ₆ + D ₂ O) δ (ppm): 1.60 (4H,
m), 3.31 (3H, s), 3.36 (6H, s), 3.47 (2)
H, m), 3.72 (2H, m), 7.00 (2H, s), 7.40 ~
7.60 (4H, m), 7.85-7.96 (2H, m). IR (KBr) ν: 1700, 1590, 1390, 1350,
1330 cm ^-1 .

Example 13 Binding of succinimidomethylene blue derivative to BSA and modified BSA Bovine serum albumin (BSA) [Miles Inc., Kankakee, Ill. USA] 50 mg, 0.5% sodium lauryl sulphate and 0.2
It was dissolved in 25 ml of 25 mM sodium phosphate buffer of pH 7 containing 5% β-mercaptoethanol. 9 this solution
Heated at 0 ° C. for 10 minutes. After cooling the reaction solution, the denatured protein was separated from the reducing agent by subjecting to Sephadex (registered trademark) G-50 column gel filtration and developing with 25 mM phosphate buffer.

All buffers used below are 0.1M phosphate buffer at pH 7 unless otherwise stated. Succinimido methylene blue derivative 1,2,3,4,5,6,
2 mg each of 7 and 8 were dissolved in 1 ml of buffer. To 0.5 ml of each solution was added 0.5 ml of buffer containing 1 mg BSA,
Incubated overnight at 6 ° C. To 0.25 ml of each solution was added 0.75 ml of a buffer containing 0.5 mg of denatured BSA, and 6
Incubated overnight at ° C. 2 in solution of succinimidomethylene blue derivative 1 incubated overnight
mg lysine was added. Sephadex for each solution
The gel was filtered through a (Sephadex) (registered trademark) G-50 column to remove unreacted methylene blue derivative. The protein concentration in each labeled solution was determined by dye-binding assay [Biorad Laboratories (Biorad Labs).
oratories), Richmond, Ca. USA]. The number of bound methylene blue derivatives was determined from the absorbance at 662 nm. Table 1 shows BSA and modified BS
Quantity of methylene blue derivative incorporated in A (MB /
BSA) and their relative chemiluminescent activity. The chemical structure of the methylene blue derivative is shown below. still,
The number of the example explaining the manufacturing example is shown in the parentheses.

[0123]

[Chemical 34]

[0124]

[Chemical 35]

[0125]

[Table 1]

Example 14 Binding of Succinimidomethylene Blue Derivatives to IgG Succinimidomethylene Blue Derivatives 2, 4, 6 and 8
2 mg of each was dissolved in 1 ml of buffer. A buffer solution containing 0.5 mg of IgG (anti-alphafetoprotein antibody A4-4, manufactured by Wako Pure Chemical Industries, Ltd., Osaka, Japan) at 0.5, 50 and 200 times in molar ratio, respectively.
Mix with ml and incubate overnight at 6 ° C. Separate each solution with Sephadex® G-5
It was subjected to gel filtration of 0 to remove unreacted methylene blue derivative. The concentrations of IgG and methylene blue derivative (MB) were determined by the method described above. These antibody-binding activities were determined by the competitive reaction between the labeled IgG-methylene blue derivative and the labeled IgG-HRP in the alpha fetoprotein ELISA method. Table 2 shows these results.

[0127]

[Table 2]

Example 15 ELIS Using Labeled IgG-Methylene Blue Derivative
A 10% solution of latex having a diameter of 3.2 μm [Sigma Chemical Co., St. Louis, Mo. US
A] 0.5 ml was washed twice with 5 ml water to prepare a carrier. The latex obtained was washed with 5 ml of 0.1 M carbonate buffer at pH 9.5 and then resuspended in 5 ml of solid buffer. After incubating this at 56 ° C for 20 minutes, 1 mg of anti-alphafetoprotein antibody (A2-95, manufactured by Wako Pure Chemical Industries, Ltd.) was added, and further at 40 ° C for 40 minutes and then at room temperature for 2 minutes.
Incubated for hours. This is 0.2% Tween-20
5 ml of 0.1 M phosphate buffer (pH 6) containing
Phosphate buffered saline containing 50% Block Ace (Snow Brand Milk Products, Sapporo, Japan) (50 mM sodium phosphate, 0.8%)
It was washed with 5 ml of sodium chloride, pH 7) and stored in phosphate buffered saline containing 25% Block Ace, 0.5% Bovine Serum Albumin, 0.02% Merthiolate.

In the ELISA method, 5 μl of an IgG-methylene blue derivative-labeled product obtained by incubating succinimidomethylene blue derivative 4 at a molar ratio of 50 times that of IgG and 250 μl of phosphate buffered saline containing 10% Block Ace, The mixture was mixed with 50 μl of the 2% latex solution prepared above, various concentrations of alpha-fetoprotein were added, and the mixture was incubated at 37 ° C. for 30 minutes. Each was washed 4 times with phosphate buffered saline containing 0.1% Tween-20 and then with phosphate buffered saline. To each of them was added 0.4 ml of 0.1 M sodium hydroxide containing 1 mM luminol, and the suspension was then added to Optically Pumped Chemiluminescence (OP
C)] method (Japanese Patent Application No. 2-249879) and transferred to a plastic tube for measurement. The result is shown in FIG. In FIG. 1, the vertical axis represents CPM (photons counted).
per minute), and the horizontal axis represents the amount of alpha fetoprotein. This result shows that the chemiluminescence amount increases linearly with the amount of alpha-fetoprotein.

Example 16 ELISA using Fab'-BSA-methylene blue derivative labeled form In this example, antibody fragment Fab'-methylene blue derivative labeled form was prepared by using bovine serum albumin as a carrier protein. 1 mg of F (ab ') ₂ prepared from anti-alpha fetoprotein antibody was added to 20 mM
Reduction was achieved by incubation with 1 ml of 0.1 M phosphate buffer, pH 6.8, containing dithiothreitol. Excess dithiothreitol was removed by gel filtration in the same buffer. The obtained protein is immediately N, N'-bis
It was reacted with 2 mg of (3-maleidopropyl) -2-hydroxy-1,3-propanediamine at 37 ° C. for 30 minutes.
Excess reagent was removed by gel filtration. According to the method described above, 1 mg of bovine serum albumin was denatured, reacted with succinimidomethylene blue derivative 4 and then reduced with dithiothreitol. The protein was recovered from gel filtration, mixed with Fab 'prepared above and incubated overnight at 6 ° C. The prepared Fab′-BSA-methylene blue derivative labeled product was prepared by the same method as described in Example 15.
It was used for the assay of alpha-fetoprotein. The result is shown in FIG. In FIG. 2, the vertical axis represents CPM and the horizontal axis represents the amount of alpha-fetoprotein. As is clear from the results, the chemiluminescence amount increases linearly with the amount of alpha-fetoprotein.

[0131]

INDUSTRIAL APPLICABILITY The photosensitizing dye derivative provided by the present invention facilitates modification of an antibody protein or the like with a photosensitizing dye, which has been difficult in the past, and a dye-catalyst labeled product which has been a problem in the modification. Can be prevented and non-specific binding to the solid surface can be prevented.

[Brief description of drawings]

FIG. 1 shows the results of the alpha-fetoprotein assay of Example 15 above.

FIG. 2 shows the results of the alpha-fetoprotein assay of Example 16 above.

Continuation of front page (51) Int.Cl. ⁵ Identification code Office reference number FI Technical display location G01N 33/533 8310-2J // A61K 31/54 ADU 7252-4C (C07D 417/12 207: 00 7019-4C 279: 00) 8314-4C

Claims (10)

[Claims] 1. A general formula: [Wherein R ₁ , R ₂ , R ₃ and R ₄ are the same or different and each is a C _1-6 alkyl group, and at least 1
Has a group that reacts with an amino group, a thiol group or a carboxyl group as a substituent, and X ⁻ represents a counter ion of phenazathionium]. 2. A general formula: [In the formula, at least one of R ₁ ′, R ₂ ′, R ₃ ′ and R ₄ ′ is a group represented by the formula: — (CH ₂ ) _m —CO—Y, and the other is a C _1-6 alkyl group. , Y represents a leaving group, m represents an integer of 1 to 6, and X ⁻ represents a counter ion of phenazathionium.]
A phenothiazine derivative represented by. 3. A general formula: [In the formula, at least one of R ₁ ′, R ₂ ′, R ₃ ′ and R ₄ ′ is a group represented by the formula: — (CH ₂ ) _m —CO—Y, and the other is a C _1-6 alkyl group. In which Y represents a leaving group, and m represents an integer of 1 to 6]. 4. The general formula: 2-amino-5-disubstituted aminophenylthio represented by the formula: wherein R ₁ ″ represents a C _1-6 alkyl group, Y represents a leaving group, and m represents an integer of 1 to 6. Sulfonic acid derivatives. 5. The general formula: [Wherein at least one of R ₁ ', R ₂ ' and R ₃ 'is of the formula:
A group represented by — (CH ₂ ) _m —CO—Y, the other represents a C _1-6 alkyl group, Y represents a leaving group, and m represents an integer of 1 to 6]. Minthiosulfonic acid derivative. 6. A general formula: [In the formula, at least one of R ₁ ′, R ₂ ′, R ₃ ′ and R ₄ ′ is a group represented by the formula: — (CH ₂ ) _m —CO—Y, and the other is a C _1-6 alkyl group. , Y represents a leaving group, m represents an integer of 1 to 6, and X ⁻ represents a counter ion of phenazathionium.]
A phenothiazine derivative represented by the formula Y is eliminated, and then a group reactive with an amino group, a thiol group or a carboxyl group is introduced: [Wherein R ₁ , R ₂ , R ₃ and R ₄ are each a C _1-6 alkyl group, at least one of which has a group reactive with an amino group, a thiol group or a carboxyl group as a substituent. , X ⁻ represents a counter ion of phenazathionium]
A method for producing a phenothiazine derivative represented by: 7. A general formula: [In the formula, at least one of R ₁ ′, R ₂ ′, R ₃ ′ and R ₄ ′ is a group represented by the formula: — (CH ₂ ) _m —CO—Y, and the other is a C _1-6 alkyl group. , Y represents a leaving group, and m represents an integer of 1 to 6], and the indamine thiosulfonic acid derivative represented by the formula [1] is oxidized with an inorganic oxidizing agent in water or an organic solvent or a mixed solvent thereof. The general formula is as follows: [In the formula, at least one of R ₁ ′, R ₂ ′, R ₃ ′ and R ₄ ′ is a group represented by the formula: — (CH ₂ ) _m —CO—Y, and the other is a C _1-6 alkyl group. , Y represents a leaving group, m represents an integer of 1 to 6, and X ⁻ represents a counter ion of phenazathionium.]
A phenothiazine derivative represented by the formula (1) is obtained, the Y is eliminated, and then a group that reacts with an amino group, a thiol group, or a carboxyl group is introduced: [Wherein R ₁ , R ₂ , R ₃ and R ₄ are each a C _1-6 alkyl group, at least one of which has a group reactive with an amino group, a thiol group or a carboxyl group as a substituent. , X ⁻ represents a counter ion of phenazathionium]
A method for producing a phenothiazine derivative represented by: 8. A general formula: [Wherein, B represents a succinimidooxycarbonyl group or a maleimido group, and m represents an integer of 1 to 6], and a triethylamine salt of an acidic amino acid is reacted with a succinimide ester derivative represented by the general formula : [Chemical formula 12] [In the formula, B represents a succinimidooxycarbonyl group or a maleimido group, m represents an integer of 1 to 6, and n represents 1
Or 2 is shown]. 9. The general formula: [Wherein R ₁ , R ₂ , R ₃ and R ₄ are the same or different and each is a C _1-6 alkyl group, and at least 1
Has a group that reacts with an amino group, a thiol group or a carboxyl group as a substituent, and X ⁻ represents a counter ion of phenazathionium] and is obtained by coupling an phenothiazine derivative to an antigen or an antibody. A method for measuring an analyte in a sample, which comprises measuring a labeled substance. 10. A general formula of an antigen or antibody coupled with its amino group, thiol group or carboxy group: [Wherein R ₁ , R ₂ , R ₃ and R ₄ are the same or different and each is a C _1-6 alkyl group, and at least 1
One having a group reactive with an amino group, a thiol group or a carboxyl group as a substituent, and X ⁻ represents a counter ion of phenazathionium], or an phenothiazine derivative represented by Labeled antibody.