JP2001089482A - Azamethine compound - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an azamethine compound having excellent water solubility, useful as a fluorescent labeled reagent and a pH indicator in the field of medical diagnosis by using absorption characteristics in an aqueous medium due to no agglutination in an aqueous solution. SOLUTION: This azamethine compound is represented by the general formula (R is a substituted or nonsubstituted alkyl group; Q is a methine group substituted with an aromatic cyclic group or a polymethine group substituted with an aromatic cyclic group; A is an oxygen atom or sulfur atom; V1, V2 and V3 are each a hydrogen atom or a monofunctional substituent and adjoining two groups among them may be mutually bonded to form a ring; M1 is a counter ion; and m is a number required to neutralize the charge of molecule).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a colorant, a light absorber,
The present invention relates to novel azamethine compounds useful as dyes for optical discs, spectral sensitizing dyes for silver halide photography and electrons, diagnostic markers, fluorescent labeling reagents, and the like.

[0002]

2. Description of the Related Art A compound that absorbs light in the visible region exhibits various colors depending on the wavelength of the absorbed light. These compounds are called dyes or dyes, and are used for coloring various materials.More advanced usage methods include dyes for optical disk materials, which are high-density information recording materials, and silver halide photographs, which are image information recording materials. It is used as a spectral sensitizing dye for electrophotography or as a filter dye. In recent years, compounds absorbing near-infrared light and infrared light have begun to be applied to plasma display filters and the like.

[0003] All of these use the absorption characteristics of dyes, but recently the emission characteristics (fluorescent or phosphorescent) of dyes have also been actively utilized. For example, fluorescent dyes can be used for antibodies, proteins, peptides, polysaccharides, nucleic acids,
In recent years, medical diagnostic techniques for labeling substances including deoxynucleic acids, derived nucleic acids, derived deoxynucleic acids, DNA fragments, RNA fragments, derived DNA fragments, derived RNA fragments, and the like by covalent bonds, ionic bonds, hydrogen bonds, etc. have become important in recent years. ing. When a methine dye is applied to such a diagnostic use, high water solubility and non-aggregation in an aqueous medium are required. In general, water-solubility can be imparted by introducing a water-soluble substituent such as a sulfo group or a carboxyl group into a dye, but the introduction of such a water-soluble substituent limits the solvent used during synthesis or As a result, a great deal of labor is required for post-treatment and purification after the reaction. Under such circumstances, development of a methine dye having high water solubility and easy synthesis operation has been demanded.

[0004]

DISCLOSURE OF THE INVENTION The present inventors have made intensive studies to solve the above-mentioned problems, and as a result, are represented by the following general formulas (I) to (III). Azamethine compounds have excellent water solubility,
Further, they have found that they do not aggregate even in an aqueous solution and can be used as a dye suitable for various uses. The present invention has been completed based on the above findings.

That is, the present invention provides a compound represented by the following general formula (I):

Embedded image (Wherein, R represents a substituted or unsubstituted alkyl group; Q represents a methine group substituted with an aromatic ring group or a polymethine group substituted with an aromatic ring group; A represents an oxygen atom or a sulfur atom; shown; V ^1, V ^2, and V ³ is a hydrogen atom or a monovalent substituent, two neighboring groups of these may be bonded to each other to form a ring; M ¹ is a counterion And m
Represents the number required to neutralize the molecular charge).

From another viewpoint, the following general formula (I)
I):

Embedded image (Wherein L ¹ , L ² , and L ^{3 each} represent a substituted or unsubstituted methine group; R ¹ and R ² each independently represent a substituted or unsubstituted alkyl group; V ¹¹ , V ¹² , V ^13, V ^14, V ^15,
And V ¹⁶ each independently represent a hydrogen atom or a monovalent substituent, of which two adjacent groups may be bonded to each other to form a ring; A ¹ and A ² each independently represent an oxygen Represents an atom or a sulfur atom; n represents an integer of 0 to 3; M ² represents a counter ion; and p represents a number necessary to neutralize the charge of the molecule). Is done.

From another viewpoint, the following general formula (II)
I):

Embedded image (Wherein, R ³ represents a substituted or unsubstituted alkyl group; M ³
Represents a counter ion; q represents a number necessary to neutralize the charge of the molecule; k represents 2 or 3).

Further, from another viewpoint, a fluorescent labeling reagent containing an azamethine compound represented by any of the above formulas (I) to (III); an azamethine compound represented by the above formula (I) to (III) A fluorescently labeled substance, preferably a diagnostic substance fluorescently labeled with an azamethine compound represented by the above general formulas (I) to (III); an azamethine compound represented by the above general formulas (I) to (III) A diagnostic drug comprising a fluorescently labeled diagnostic substance;
A method of diagnosing using a diagnostic substance fluorescently labeled with an azamethine compound represented by any one of the above general formulas (I) to (III); The use of an azamethine compound of formula (I) is provided by the present invention.

[0009]

DETAILED DESCRIPTION OF THE INVENTION In the present specification, the alkyl moiety of an alkyl group or a substituent containing an alkyl moiety (for example, an alkoxy group) may be any of linear, branched, cyclic, or a combination thereof. The number of carbon atoms is about 1 to 20 unless otherwise specified. R, R
Examples of the alkyl group represented by ¹ , R ² and R ³ include, for example,
Methyl group, ethyl group, n-propyl group, isopropyl group, cyclopropyl group, n-butyl, sec-butyl group, tert-butyl group, cyclopropylmethyl group, n
-Pentyl, n-hexyl, cyclohexyl and the like. The alkyl groups represented by R ¹ and R ² may be the same or different. R, R ¹ , R ² , and R ³
May have one or more substituents at any position on the alkyl chain. When it has two or more substituents, they may be the same or different.

The type of the substituent on the alkyl group represented by R, R ¹ , R ² and R ³ is not particularly limited. For example, in order to introduce the compound of the present invention as a fluorescent label into a substance to be labeled, It is preferable that a reactive substituent capable of forming a bond such as a covalent bond, an ionic bond, or a hydrogen bond with a labeling substance is introduced ("reactive substituent" in the present specification).
Means a substituent having the above characteristics). R, R
Examples of the reactive substituent that can be introduced on the alkyl group represented by ¹ , R ² and R ³ include a succinimidyl ester group, a halogen-substituted triazinyl group, a halogen-substituted pyrimidinyl group, a sulfonyl halide group, an α-haloacetyl group, Maleimidyl group, aziridinyl group and the like can be mentioned.

In addition to these reactive substituents, for example, a halogen atom (in the present specification, a “halogen atom” may be any of a fluorine, chlorine, bromine or iodine atom), a mercapto group , Cyano group,
Nitro group, carboxyl group, phosphate group, sulfo group, hydroxy group, amino group, isothiocyanate group, isocyanate group, alkoxy group having 1 to 8 carbon atoms (for example, methoxy group, ethoxy group, etc.), carbon 6 or more atoms
20 aryloxy groups (e.g., phenoxy group, naphthoxy group, etc.), alkoxycarbonyl groups having 2 to 10 carbon atoms (e.g., methoxycarbonyl group, ethoxycarbonyl group), aryloxycarbonyl having 6 to 20 carbon atoms Group (eg, phenoxycarbonyl group), acyl group having 2 to 10 carbon atoms (eg, acetyl group, pivaloyl group, etc.), acyloxy group having 2 to 8 carbon atoms (eg, acetyloxy group, benzoyloxy) Group), an acylamino group having 2 to 8 carbon atoms (eg, an acetylamino group), and a carbon atom having 1 to 8 carbon atoms.
8 sulfonyl groups (eg, methanesulfonyl group, ethanesulfonyl group, benzenesulfonyl group, etc.), sulfinyl groups having 1 to 20 carbon atoms (eg, methanesulfinyl group, ethanesulfinyl group, benzenesulfinyl group, etc.), carbon atom A sulfonylamino group having 1 to 8 carbon atoms (for example, methanesulfonylamino group, ethanesulfonylamino group, benzenesulfonylamino group, etc.); a carbamoyl group having 1 to 10 carbon atoms (for example, carbamoyl group, methylcarbamoyl group, A substituted amino group having 1 to 20 carbon atoms (eg, a methylamino group, a dimethylamino group, a benzylamino group, an anilino group, a diphenylamino group, etc.);
A sulfamoyl group having 2 to 10 carbon atoms (for example, a methylsulfamoyl group, an ethylsulfamoyl group, a piperidinosulfamoyl group, etc.), and a carbon atom number of 0 to 15
(E.g., trimethylammonium group, triethylammonium group, etc.) having 0 carbon atoms
To 15 hydrazino groups (e.g., trimethylhydrazino group, etc.), ureido groups having 1 to 15 carbon atoms (e.g., ureido groups, N, N-dimethylureido groups, etc.), and imides having 1 to 15 carbon atoms. Group (e.g., succinimide group), alkylthio group having 1 to 20 carbon atoms (e.g., methylthio group, ethylthio group, etc.), arylthio group having 6 to 20 carbon atoms (e.g., phenylthio group, p-methylphenyl) A thio group, a p-chlorophenylthio group, a 2-pyridylthio group, a naphthylthio group, etc., a substituted or unsubstituted heterocyclic group having 1 to 20 carbon atoms (for example, a pyridyl group, a 5-methylpyridyl group, a thienyl group,
Furyl, morpholino, tetrahydrofuryl, 2-
A pyrazyl group), an unsaturated hydrocarbon group having 2 to 18 carbon atoms (for example, a vinyl group, an ethynyl group, a 1-cyclohexenyl group, a benzylidine group, a benzylidene group, etc.), a substituted or unsubstituted group having 6 to 20 carbon atoms. Unsubstituted aryl group (for example, phenyl group, 4-sulfophenyl group, 2,5
-Disulfophenyl group, 4-carboxyphenyl group, naphthyl group, etc.) and an alkyl group having 1 to 20 carbon atoms (eg, methyl group, ethyl group, propyl group, etc.). These groups may be further substituted with the groups described above (for example, a sulfopropyl group, a sulfobutyloxy group, a carboxybutyroxycarbonyl group, etc.). Further, the above-mentioned phosphate group, carboxyl group or sulfo group may be in a salt state.

Preferred examples of R, R ¹ , R ² and R ³ include an unsubstituted alkyl group having 2 to 10 carbon atoms, a sulfo group, a phosphoric acid group, a carboxyl group, an isothiocyanate group and a succinimidyl ester. Group, a sulfonyl halide group, an α-haloacetyl group, or an alkyl group having 2 to 10 carbon atoms substituted with a maleimidyl group, more preferably an unsubstituted alkyl group having 2 to 6 carbon atoms, or a sulfo group. A C2-C6 alkyl group substituted by a group, a carboxyl group, an isothiocyanate group or a succinimidyl ester group.

In the azamethine compound represented by the general formula (I), Q represents a methine group substituted with an aromatic ring group or a polymethine group substituted with an aromatic ring group. As the aromatic ring group, a 5- to 16-membered ring group, preferably a 5- to 14-membered ring group containing at least one aromatic ring can be used. The aromatic ring group is a 2- to 3-cyclic fused aromatic ring group or 1
Alternatively, a 2- to 3-cyclic group containing two or more partially saturated rings may be used. The aromatic ring group may contain one or more hetero atoms such as an oxygen atom, a nitrogen atom, and a sulfur atom as ring-constituting atoms. R can be located at any position on the ring to be formed.
May be substituted with one or more substituents (including reactive substituents) or alkyl groups exemplified as substituents on the alkyl group. When two or more substituents are present, they may be the same or different. Although the number of carbon atoms constituting polymethine is not particularly limited, for example, 3 to
Seven, more preferably about three to five.

More specifically, Q is a methine dye (comprising a methine group or a polymethine group and two identical or different heterocyclic groups or aromatic groups bonded to both ends of the methine group or the polymethine group). ) Is preferably a partial structure excluding one heterocyclic group or aromatic group from the above. The type of the methine dye is not particularly limited, and examples thereof include a cyanine dye, a merocyanine dye, a rhodocyanine dye, a trinuclear merocyanine dye, an allopolar dye, a hemicyanine dye, and a styryl dye. For details of these dyes, see, for example, FM Hammer.
(FM Harmer), Heterocyclic Compounds-Cyanine Dyes and Relat
ed Compounds) ", John Wiley and Sons
(John Wiley and Sons)-New York, London, 1
964, DM Sturmer, Heterocyclic Compounds-Special Topics in Heterocyclic Chemistry (H
eterocyclic Compounds? Special Topics in Heteroc
yclic Chemistry), Chapter 18, Section 14, 482 to 51
Page 5, John Wiley and Sons
and Sons)-New York, London, 1977, etc. Cyanine dyes, merocyanine dyes and rhodacyanine dyes include those described in US Pat. No. 5,340,69.
No. 4, pp. 21 and 22, Formula (XI), Formula (XII), and Formula (XII)
The dyes represented by I) are preferred. The compound represented by the general formula (I) is more preferably the case where the compound represented by the general formula (I) has a cyanine dye structure represented by the general formula (II).

V ¹ , V ² , and V ³ , or V ¹¹ , V ¹² , V
^13, V ^14, V ^15, and the type of the monovalent substituent represented by V ¹⁶ is not particularly limited, they may be the same or different. As the substituent represented by these groups, for example, those exemplified as the substituent on the alkyl group represented by R (including a reactive substituent) or an alkyl group can be used. V
^1, two adjacent groups of the adjacent two groups or ^{V 11, V 12, V 13} , V 14, V 15, and V ^16, of V ^2, and V ³ are connected to each other It may form a saturated or unsaturated ring. Examples of the ring formed include a 5- to 7-membered ring. The unsaturated ring may form a condensed aromatic ring. The unsaturated ring may contain a hetero atom such as an oxygen atom, a nitrogen atom and a sulfur atom. At any position on the formed ring, one or more alkyl groups may be substituted with the substituents exemplified as the substituents on the alkyl group represented by R.

V ¹ , V ² , V ³ , V ¹¹ , V ¹² , V ¹³ ,
Preferred examples of V ¹⁴ , V ¹⁵ , and V ¹⁶ include, for example,
Hydrogen atom, halogen atom, carboxyl group, sulfo group,
Alkyl group, alkoxy group, isothiocyanate group, isocyanate group, succinimidyl ester group, halogen-substituted triazinyl group, halogen-substituted pyrimidinyl group,
A sulfonyl halide group, an α-haloacetyl group, a maleimidyl group and an aziridinyl group, more preferably a hydrogen atom or a halogen atom.

M ¹ , M ² or M ³ represents a counter ion,
It may be either a cation or an anion. The cation may be either an inorganic cation or an organic cation, and examples thereof include alkali metal ions such as sodium ion, potassium ion and lithium ion, and organic ions such as tetraalkylammonium ion and pyridinium ion. The anion may be either an inorganic anion or an organic anion, and a halogen anion (for example,
Fluorine ion, chlorine ion, bromine ion, iodine ion, etc., substituted aryl sulfonate ion (for example, p-
Toluenesulfonate ion, p-chlorobenzenesulfonate ion, etc., aryldisulfonate ions (e.g., 1,3-benzenedisulfonate ion, 1,5-naphthalenedisulfonic acid ion, etc.), alkyl sulfate ions (e.g., methyl sulfate) Ion), sulfate ion, thiocyanate ion, perchlorate ion, tetrafluoroborate ion, picrate ion, acetate ion, trifluoromethanesulfonate ion and the like. Further, M ¹ , M ² , or M ³ may be a hydrogen ion.
Examples of preferred counter ions include, for example, ammonium ion, alkali metal ion, halogen anion, substituted aryl sulfonate ion and the like, more preferably alkali metal ion, halogen anion,
Substituted aryl sulfonate ions can be mentioned.
m, p, or q each represents a number necessary to neutralize the charge of the molecule, and when the compound represented by the formula (I) to the formula (III) is a nonion or betaine (an intramolecular counter ion), In some cases, m, p, or q may be 0.

L ¹ , L ² and L ³ each independently represent a substituted or unsubstituted methine group. The number, position and type of substituents on the methine group are not particularly limited, and when two or more substituents are present, they may be the same or different. Examples of the substituent include a substituted or unsubstituted carbon atom having 1 to 15, preferably 1 carbon atom.
Alkyl groups having 1 to 5 carbon atoms (e.g., methyl group, ethyl group, carboxyethyl group, etc.), substituted or unsubstituted carbon atoms having 6 to 30, preferably carbon atoms having 1 to 5 carbon atoms. An aryl group having 6 to 20, more preferably 6 to 15 carbon atoms (for example, phenyl group, o
-Carboxyphenyl group), substituted or unsubstituted carbon atoms having 3 to 20, preferably 4 to 15 carbon atoms,
More preferably, a heterocyclic group having 6 to 10 carbon atoms (eg, N, N-dimethylbarbituric acid group and the like),
A halogen atom having 1 to 20 carbon atoms, preferably 1 to
15, more preferably 1 to 10 alkoxy groups (eg, methoxy group, ethoxy group, etc.), 0 to 0 carbon atoms
20, preferably an amino group having 2 to 15 carbon atoms, more preferably 4 to 15 carbon atoms (eg, a methylamino group, a dimethylamino group, an N-methyl-N-phenylamino group, an N-methylpiperazino group) Etc.), an alkylthio group having 1 to 15 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 8 carbon atoms (for example, methylthio group, ethylthio group, etc.), and 6 carbon atoms. To 20, preferably 6 to 18 carbon atoms, more preferably 6 to 15 carbon atoms such as an arylthio group (e.g., phenylthio group, p-methylthio group). ^Two substituents on L ¹ , L ² and L ³ may be bonded to each other to form a ring. For example, ^two substituents on L ² and L ³ may be bonded to each other to form an alkylene chain having 2 or 3 carbon atoms to form a ring.

N represents 1, 2 or 3, and preferably 1 or 2. In particular, when n is 1, the efficiency of excitation by inexpensive helium-neon laser excitation (excitation wavelength: 633 nm) becomes extremely high, so that n is most preferably 1. k represents 2 or 3.

The compounds of the present invention represented by the above general formulas (I) to (III) may exist as hydrates or solvates, and these substances are all included in the scope of the present invention. You. Further, the compound of the present invention may have one or more asymmetric carbons depending on the type of the substituent. However, stereoisomers such as optical isomers and diastereoisomers, a mixture of stereoisomers, a racemate, and the like Are all included in the scope of the present invention.

Preferred examples of the compounds of the present invention represented by the above general formulas (I) to (III) are shown below, but the scope of the present invention is not limited to the following specific compounds.

[0022]

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[0023]

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[0024]

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[0025]

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[0026]

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[0027]

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[0028]

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[0029]

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[0030]

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[0031]

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Since the method of producing a representative compound is specifically shown in the examples of the present specification, those skilled in the art may appropriately refer to the specific description of the following examples to determine the starting compounds, reaction conditions, reagents and the like. The above formula (I) can be selected and optionally modified or modified in the methods described in the examples.
It is possible to produce any compound included in (III) to (III). However, the method for producing the compounds of the present invention represented by the above general formulas (I) to (III) is not particularly limited, and it goes without saying that compounds produced by any method are included in the scope of the present invention.

The applications of the compounds of the present invention represented by the above general formulas (I) to (III) are not particularly limited, and include colorants, light absorbers, dyes for optical disks, silver halide photography and spectral sensitization of electrons. It can be used as a dye for various uses such as a dye, a diagnostic marker, a fluorescent labeling reagent, and a pH indicator. Of these, fluorescently labeled dyes are a preferred use of the compounds of the present invention. The substance to be labeled that can be labeled with the compound of the present invention is not particularly limited, and any substance such as a low-molecular compound, a high-molecular compound, an organic compound, an inorganic compound, a biological substance, a natural organic compound, or a microorganism can be used as a target for labeling. It is possible. More specifically, antibodies, proteins, peptides,
Enzyme substrate, hormone, lymphokine, metabolite, receptor, antigen, hapten, lectin, avidin, streptavidin, toxin, carbohydrate, polysaccharide, nucleic acid, deoxynucleic acid, derived nucleic acid, derived deoxynucleic acid, DNA fragment, RNA fragment, derived DNA Fragments, derived RNA fragments, natural drugs, viral particles, bacterial particles, viral components, yeast components, blood cells, blood cell components, bacteria, bacterial components,
Natural or unnatural lipids, synthetic drugs, poisons, environmental pollutants,
Polymer, polymer particles, glass particles, plastic particles,
A polymer film or the like can be mentioned as the substance to be labeled.

Among these, preferred labeling substances include antibodies, proteins, peptides, nucleotides, hormones,
Sugars, lipids, vitamins, alkaloids, antibiotics, etc. Specific examples of proteins or peptides, for example, IgG, IgA, IgM, IgD, immunoglobulins such as IgE, monoclonal antibodies against various proteins and membrane antigens of leukocytes, peroxidase, glucose oxidase,
Enzymes such as alkaline phosphatase can be mentioned,
Specific examples of nucleotides include, for example, DNA, RNA, synthetic oligonucleotides, synthetic polynucleotides, ATP, C
TP, GTP, TTP, UTP, dATP, dCTP, dGTP, dTTP, dUTP, d
Examples thereof include dATP, ddCTP, ddGTP, ddTTP, ddUTP, and derivatives thereof. Specific examples of sugars include
For example, glycogen, starch, in addition to polysaccharides such as mannan, oligosaccharides and glucose, monosaccharides such as mannose and the like, and examples of lipids include, for example, phosphatidylcholine, phosphatidylethanolamine,
Fats, fatty acids and the like can be mentioned. Examples of hormones include insulin, growth hormone, epidermal growth factor, oxytocin, vasopressin, peptide hormones such as secretin, androgens, steroid hormones such as estrogen, adrenaline, catecholamines such as noradrenaline, and the like.
As vitamins, for example, vitamin A, vitamin B
(B1, B2, B6, B12 etc.), Vitamin C, Vitamin D,
Vitamin E, biotin, folic acid and the like can be mentioned. Examples of the alkaloids include opium alkaloids, tropane alkaloids such as atropine, indole alkaloids such as vinblastine, and isoquinoline alkaloids such as spinach, and examples of the antibiotics include penicillin, cephalosporin, kanamycin, and erythromycin. Can be. Among these labeled substances, a substance that can be used for diagnosis (hereinafter referred to as a “diagnostic substance”.
Antibodies, proteins, peptides, etc.) are preferred labeling substances.

There are various known techniques for introducing a fluorescent labeling reagent into a substance to be labeled. When the compound of the present invention is used as a fluorescent labeling reagent, a means available to those skilled in the art is appropriately selected and used. It is possible to For example, a functional group such as an amino group or a hydroxyl group in the substance to be labeled is directly bonded to a reactive substituent such as a carboxyl group or an active ester group in the compound of the present invention by ionic bonding or covalent bonding, or The compound of the present invention may be reacted after chemical modification such as introduction of a linker into a part of the labeling substance. The labeled substance after the reaction can be purified by a general-purpose separation technique such as chromatography, electrophoresis, and recrystallization.

When the compound of the present invention is used for DNA analysis, for example, Ruth (Jerry L. Ruth, DNA, 3, 123)
(1984)), the compound of the present invention can be incorporated into a probe or a primer. A method of labeling by reacting a hydrophilic polyfunctional polymer such as a protein with a dye compound having a reactive substituent (eg, active ester, isothiocyanate, iodoacetyl, etc.) is described in, for example, US Pat. No. 5,569,587. It can be easily carried out according to the method described in the book. The dye having a carboxyl group that is not active esterified can be labeled, for example, according to the method described in JP-A-6-222059.

For example, by labeling an anti-tumor antibody with the compound of the present invention and bringing the labeled antibody into contact with a tissue or an organ, the presence of cancer cells or cancer tissue can be proved. For diagnosis, tissue sections and the like may be fixed under an appropriate method such as a paraffin method and observed under a microscope.
A living tissue can also be immunochemically stained and observed using an endoscope. Recently, various fluorescent imaging methods using near-infrared fluorescent materials have been proposed (for example,
JP-A-9-309845; J. Neurosurg., 87, p.
p.738-745, 1997; Medical Electronics and Biotechnology, 34, pp.316-32
2, 1996), the compounds of the present invention can be used as diagnostics using fluorescent imaging techniques.

When a diagnostic substance (for example, a labeled antibody) labeled with the compound of the present invention is used as a pharmaceutical for diagnosis, a pharmaceutical composition is prepared using one or more additives for pharmaceutical preparations. It is preferable to prepare as a form. For example, a pharmaceutical composition in the form of a solid or a solution can be prepared using appropriate pharmaceutical additives such as a buffer, a solubilizer, a pH adjuster, an excipient, and a preservative. The form of a pharmaceutical composition suitable for diagnosis or treatment and a method for producing the same,
A person skilled in the art can select as appropriate.

[0039]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to Examples, but the scope of the present invention is not limited to the following Examples. The compound numbers in the examples correspond to the compound numbers shown as the preferred compounds above. Example 1: Synthesis of compound I-1

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(Synthesis of Compound I-1b) Triethyl orthoacetate (40 ml) and p-toluenesulfonic acid monohydrate (30 mg) were added to 2-amino-3-hydroxypyridine (5.4 g, 49 mmol).
Was added and reacted at 180 ° C. for 2 hours. Excess orthoester was distilled off under reduced pressure, and the residue was purified by a silica gel column (eluent: ethyl acetate), and then recrystallized from hexane to obtain a compound I-1b. Yield: 5.3 g, Yield: 81% H-NMR (CDCl ₃ ), δ; 8.52 (dd, 1H), 7.77 (dd, 1H), 7.25
(dd, 1H), 2.73 (s, 3H) Mass (Posi): 135 (M + H)

(Synthesis of Compound I-1a) Compound I-1b (1.34
g, 10 mmol) was dissolved in acetone (10 ml), methyl iodide (1.6 g, 11 mmol) was added, and the mixture was heated under reflux for 45 minutes.
When ethyl acetate (20 ml) was added to the reaction mixture, yellow crystals of I-1a precipitated. Yield: 1.7 g, yield: 62% H-NMR (CDCl ₃ ), δ; 9.50 (d, 1H), 8.82 (d, 1H), 8.16 (d
d, 1H), 4.70 (s, 3H), 2.95 (s, 3H) Mass (Posi): 149 (M)

(Synthesis of Compound I-1) Compound I-1a (1.0 g,
3.6 mmol) was dissolved in N, N-dimethylacetamide (3 ml), and triethyl orthoformate (1.07 g, 7.2 mmol) was added and reacted at 130 ° C. for 15 minutes. When the reaction solution was cooled with ice, an iodine salt of Compound I-1 was precipitated. The iodine salt taken out by filtration was dissolved in methanol, and tetrabutylammonium perchlorate (1.3 g) was added, whereby crude crystals of compound I-1 were precipitated. The crude crystals were recrystallized from methanol to obtain compound I-1. Yield: 0.95 g, yield: 65% Absorption maximum (water): 569 nm Molecular extinction coefficient: 140,000 mass (posi): 307 (M-ClO ₄ )

Example 2: Synthesis of compound II-1

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(Synthesis of Compound II-1b) 2-amino-3-
Hydroxypyridine (5.5 g, 50 mmol) and dimethyl adipimidate dihydrochloride (6.1 g, 25 mmol) were added to ethanol (100 g).
ml) and heated under reflux for 9 hours. The reaction solution was concentrated under reduced pressure, saturated aqueous sodium hydrogen carbonate was added, and the mixture was extracted with ethyl acetate. The ethyl acetate layer was concentrated under reduced pressure to obtain compound II-1b as crystals. Yield: 3.4 g, Yield: 46% H-NMR (CDCl ₃ ), δ; 8.53 (d, 2H), 7.77 (d, 2H), 7.26 (d
d, 2H), 3.10 (t, 4H), 2.13 (m, 4H) Mass (Posi): 295 (M + H)

(Synthesis of Compound II-1) Compound II-1b (0.59
g, 2 mmol) in acetone (6 ml).
(0.38 ml, 4 mmol) was added and reacted at 60 ° C. for 2 hours. Compound II is obtained by adding ethyl acetate and hexane to the reaction mixture.
-1a precipitated as an oily residue. After separating the oil by decantation, triethyl orthoformate (0.5 ml) and N, N-dimethylacetamide (5 ml) were added to the oil and reacted at 150 ° C. for 1 hour. When the reaction solution was cooled with ice, a sulfate of compound II-1 was precipitated. The sulfate taken out by filtration was dissolved in methanol, and tetrabutylammonium perchlorate was added to precipitate crystals of compound II-1. Yield: 0.3 g, Yield: 35% H-NMR (DMSO-d ₆ ), δ; 8.06 (d, 2H), 7.99 (m, 1H), 7.87
(d, 2H), 7.13 (m, 2H), 4.07 (s, 6H), 2.98 (s, 4H) Mass (Posi): 333 (M-ClO ₄ ) Maximum absorption (water): 643 nm Molecular absorption coefficient: 155000

Example 3 Synthesis of Compound III-1

Embedded image (Synthesis of Compound III-1a) 2-amino-3-hydroxypyridine (5.5 g, 50 mmol) and dimethylpimelimidate.2
Hydrochloride (6.4 g, 25 mmol) was dissolved in ethanol (50 ml), and the mixture was heated under reflux for 10 hours. The reaction solution was concentrated under reduced pressure, saturated aqueous sodium hydrogen carbonate was added, and the mixture was extracted with ethyl acetate. The ethyl acetate layer was concentrated under reduced pressure to obtain compound III-1a as crystals. Yield: 3.5 g, Yield: 45% H-NMR (DMSO-d ₆ ), δ; 8.51 (dd, 2H), 8.12 (d, 2H), 7.4
3 (dd, 2H), 3.05 (t, 4H), 1.92 (m, 4H), 1.59 (m, 2H) Mass (Posi): 309 (M + H)

(Synthesis of Compound III-1) Compound I shown in Example 2
The reaction was carried out on the same scale and under the same conditions as for I-1.
I got Yield: 0.36 g, Yield: 40% H-NMR (DMSO-d ₆ ), δ; 8.40 (m, 1H), 8.10 (d, 2H), 7.93
(m, 2H), 7.14 (m, 2H), 4.07 (s, 6H), 2.63 (t, 4H), 1.86
(m, 2H) Mass (Posi): 347 (M-ClO ₄ ) Maximum absorption (water): 612 nm Molecular absorption coefficient: 150,000

Example 4: Synthesis of compound VI-1

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(Synthesis of Compound VI-1d) 2-Amino-3,5
-Dichloropyridine (20 g, 0.12 mol) and acetic anhydride (12.6
g) and heated under reflux for 1 hour. When the reaction solution was cooled to room temperature, compound VI-1d was precipitated. The crystals were recrystallized from a mixed solvent of ethyl acetate and hexane to obtain Compound VI-1d. Yield: 24 g, Yield: 95% H-NMR (CDCl ₃ ), δ; 8.27 (d, 1H), 7.81 (bs, 1H), 7.73
(d, 1H), 2.48 (s, 3H) Mass (Posi): 206 (M + H)

(Synthesis of Compound VI-1c) Compound VI-1d (15.0
g, 73 mmol) was dissolved in toluene (800 ml), Lawesson's reagent (14.8 g, 36.6 mmol) was added, and the mixture was reacted at 100 ° C. for 3 hours. The residue obtained by concentrating the reaction solution was purified by a silica gel column (eluent: ethyl acetate / hexane = 1/1), and further recrystallized from a mixed solvent of ethyl acetate and hexane to obtain a compound VI-1c. Yield: 15.2 g, yield: 94% mass (posi): 222 (M + H)

(Synthesis of Compound VI-1b) Compound VI-1c (22.1
g, 0.10 mol) in dioxane (150 ml), and sodium hydride (60% in oil, 4.0 g, 0.10 mol) was added.
The mixture was heated under reflux for an hour. The reaction mixture was concentrated under reduced pressure, extracted with chloroform, and the residue obtained by concentrating the chloroform layer under reduced pressure was subjected to an alumina column (eluent: hexane / ethyl acetate = 3/3).
Purification in 1) gave compound VI-1b. Yield: 11.8 g, Yield: 64% H-NMR (CDCl3), δ; 8.64 (d, 1H), 8.17 (d, 1H), 2.91
(s, 3H) Mass (Posi): 185 (M + H)

(Synthesis of Compound VI-1a) Compound VI-1b (3.0
g, 16 mmol) was dissolved in acetone (50 ml), methyl iodide (2.5 g, 18 mmol) was added, and the mixture was heated under reflux for 6 hours.
When ethyl acetate (20 ml) was added to the reaction solution, yellow crystals of VI-1a precipitated. Yield: 4.6 g, 87% H-NMR (DMSO-d ₆ ), δ; 9.58 (d, 1H), 9.45 (d, 1H), 4.57
(s, 3H), 3.14 (s, 3H) mass (posi): 199 (M)

(Synthesis of Compound VI-1) Compound VI-1a (1.5 g,
4.6 mmol) was added to a mixed solvent of pyridine (5 ml) and acetic acid (3 ml), and triethyl orthoformate (2 ml) and triethylamine (0.6 ml) were added and reacted at 120 ° C. for 2 hours. The crude crystals precipitated when ethyl acetate was added to the reaction solution were filtered out. Recrystallization from a mixed solvent of chloroform and methanol was repeated twice to obtain compound VI-1. mass (posi): 408 (M-ClO ₄ ) Maximum absorption (water): 651 nm Molecular absorption coefficient: 120,000

Example 5: Evaluation of water solubility and aggregability 1.0 × 10 ⁻³ mol of the compound of the present invention and a dye for comparison
An aqueous solution having a concentration of / l and a 1.0 M aqueous sodium chloride solution were prepared, and the water solubility and cohesiveness were evaluated. Table 1 shows the results.

[0055]

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[0056]

[Table 1]

From Table 1, it is apparent that the compound of the present invention has high water solubility. Further, when the absorption spectrum shape of the compound of the present invention was evaluated in an aqueous solution and a 1.0 M aqueous solution of sodium chloride, both compounds were evaluated. According to the shape in the organic solvent, it became clear that no aggregation occurred.

[0058]

The azamethine dye of the present invention can be present as a monomer in an aqueous medium without introducing a water-soluble group such as a sulfo group or an aggregation-inhibiting group, which makes the synthesis operation complicated. The azamethine dye of the present invention can be applied as a fluorescent labeling reagent or a pH indicator in the field of medical diagnosis by utilizing absorption characteristics in an aqueous medium.

Claims (4)

[Claims] 1. The following general formula (I): (Wherein, R represents a substituted or unsubstituted alkyl group; Q represents a methine group substituted with an aromatic ring group or a polymethine group substituted with an aromatic ring group; A represents an oxygen atom or a sulfur atom; shown; V ^1, V ^2, and V ³ is a hydrogen atom or a monovalent substituent, two neighboring groups of these may be bonded to each other to form a ring; M ¹ is a counterion And m
Represents the number required to neutralize the molecular charge). 2. The following general formula (II): (Wherein L ¹ , L ² , and L ^{3 each} represent a substituted or unsubstituted methine group; R ¹ and R ² each independently represent a substituted or unsubstituted alkyl group; V ¹¹ , V ¹² , V ^13, V ^14, V ^15,
And V ¹⁶ each independently represent a hydrogen atom or a monovalent substituent, of which two adjacent groups may be bonded to each other to form a ring; A ¹ and A ² each independently represent an oxygen A represents an atom or a sulfur atom; n represents an integer of 0 to 3; M ² represents a counter ion; and p represents a number necessary to neutralize the electric charge of the molecule). 3. The following general formula (III): (Wherein, R ³ represents a substituted or unsubstituted alkyl group; M ³
Represents a counter ion; q represents a number necessary to neutralize the charge of the molecule; k represents 2 or 3). A fluorescent labeling reagent comprising the azamethine compound according to any one of claims 1 to 3.