JP2003064083A - 2,3,3-TRIMETHYL-3H-PYRROLO[2,3-b]PYRIDINE-5-SULFONIC ACID AND DERIVATIVE THEREOF - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an azacyanine coloring matter having high fluorescence intensity and high water solubility. SOLUTION: This compound is represented by general formula (I) or general formula (II) (M is a hydrogen atom or a countercation of sulfonic acid anion in the general formula (I); R is a <=10C substituted or nonsubstituted alkyl group in the general formula (II)) or its salt.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a fluorescent dye. The present invention also relates to compounds useful as intermediates for the production of fluorescent dyes.

[0002]

2. Description of the Related Art Fluorescent dyes have been known for a long time, but they have attracted attention as markers for labeling in the fields of biochemistry and genetic diagnosis in recent years, and in this field, they are extremely important as fluorescent labeling technology. Has arrived. Fluorescein dye derivatives, rhodamine dye derivatives and the like are known as fluorescent dyes for this purpose, and in addition to these, cyanine dyes Cy3 and Cy5 manufactured by Amersham are widely used as labeling fluorescent dyes. However, since the excitation wavelengths of these dyes are limited to two types of wavelengths, Cy3 and Cy5, it is desired to support a wider range of excitation wavelengths in order to expand the applications, and further increase the fluorescence intensity. It is also strongly desired from the viewpoint of improving the sensitivity of labeling.

On the other hand, Journal of Chemical Society (JCS), 1959, p.
It has been reported that an azacyanine dye derived from a pyrrolo [2,3-b] pyridine compound exhibits strong fluorescence. However, there are few known derivatives of this dye, and it is hard to say that it can respond to various excitation wavelengths. The 3H-pyrrolo [2,3-b] pyridine compound, which is a synthetic intermediate of the dye, is important for the purpose of coping with various excitation wavelengths. Also, since many fluorescent dyes are used as labels for biological substances, it is important that they have a high solubility in water. From this point as well, a sulfo group is introduced into a 3H-pyrrolo [2,3-b] pyridine compound. Therefore, a compound having improved water solubility has been strongly desired.

[0004]

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention and Problems to be Solved by the Invention An object of the present invention is to provide an azacyanine dye having high fluorescence intensity and high water solubility. Also,
Another object of the present invention is to provide a 3H-pyrrolo [2,3] having a sulfo group introduced, which is an intermediate for producing the azacyanine dye.
-B] to provide a pyridine compound. The present inventors have conducted extensive studies to solve the above problems, and succeeded in providing a compound represented by the following general formula.

That is, the present invention provides the following general formula (I) or general formula (II):

[Chemical 3] (In the general formula (I), M represents a hydrogen atom or a counter cation of a sulfonate anion;
Represents a substituted or unsubstituted alkyl group having 10 or less carbon atoms) or a salt thereof.

From another point of view, according to the present invention, the following general formula (III) or (IV):

[Chemical 4] (In the formula, R ¹ , R ² , R ³ , and R ⁴ each independently represent a substituted or unsubstituted alkyl group having 10 or less carbon atoms; Y ¹
And Y ² are each independently a hydrogen atom, a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, a substituted or unsubstituted alkenyl group having 2 to 6 carbon atoms, a substituted or unsubstituted alkynyl group having 2 to 6 carbon atoms. Group, substituted or unsubstituted alkoxy group having 1 to 6 carbon atoms, substituted or unsubstituted aryl group having 6 to 10 carbon atoms, substituted or unsubstituted aryloxy group having 6 to 10 carbon atoms, sulfo group, cyano group A halogen atom, a carboxyl group, a substituted or unsubstituted alkoxycarbonyl group having 1 to 6 carbon atoms, a substituted or unsubstituted carbamoyl group having 1 to 6 carbon atoms, a substituted or unsubstituted amino group having 0 to 6 carbon atoms, A substituted or unsubstituted sulfonyl group having 0 to 10 carbon atoms, a substituted or unsubstituted sulfamoyl group having 0 to 10 carbon atoms, a nitro group, and 1 carbon atom
~ 6 substituted or unsubstituted carboxamide group, carbon number 1
~ 6 substituted or unsubstituted sulfonamide group, 1 to 6 carbon substituted or unsubstituted ureido group, 2 to 2 carbon atoms
6-substituted or unsubstituted alkoxycarbonylamino group, 1- to 6-carbon substituted or unsubstituted alkylthio group, 6- to 10-carbon substituted or unsubstituted arylthio group, 1- to 6-carbon substituted or unsubstituted An aromatic heterocyclic thio group or a hydroxyl group, n is 0 or 1)
A compound represented by or a salt thereof is provided.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The type of the counter cation of sulfonic acid represented by M is not particularly limited, and examples thereof include ammonium ion (wherein ammonium ion is an unsubstituted ammonium ion, a proton adduct of alkylamine or arylamine, Includes quaternary ammonium ions), metal cations (lithium, sodium, potassium, magnesium,
(Ions of metals such as calcium, zinc, iron, and silver), sulfonium ions, phosphonium ions, and the like.

In the present specification, the term "substituted or unsubstituted" for a functional group means that one or more substituents may be present on the functional group. In such a case, the type and number of substituents and the substitution position are not particularly limited. As the substituent, for example, a halogen atom (for example, a chlorine atom, a bromine atom, an iodine atom, etc.), an alkyl group (a linear, branched, cyclic, or an alkyl group consisting of a combination thereof is meant. The alkyl group may be substituted or unsubstituted. Straight-chain or branched-chain substituted or unsubstituted alkyl group (preferably straight-chain or branched-chain substituted or unsubstituted alkyl group having 1 to 30 carbon atoms, for example, methyl group, ethyl group, n
-Propyl group, isopropyl group, t-butyl group, n-octyl group, eicosyl group, 2-chloroethyl group, 2-cyanoethyl group, 2-ethylhexyl group, etc., substituted or unsubstituted cycloalkyl group (preferably carbon) Number of atoms 3
To 30 substituted or unsubstituted cycloalkyl groups such as cyclohexyl, cyclopentyl, 4-n-dodecylcyclohexyl, etc., substituted or unsubstituted bicycloalkyl groups (preferably substituted with 5 to 30 carbon atoms). Or an unsubstituted bicycloalkyl group, that is, a monovalent residue obtained by removing one hydrogen atom from a bicycloalkane having 5 to 30 carbon atoms, for example, bicyclo [1,
2,2] heptan-2-yl group, bicyclo [2,2,2]
2] octane-3-yl group) and further substituted or unsubstituted tricycloalkyl groups having many ring structures. The alkyl moiety in each substituent described below (for example, an alkyl group of an alkylthio group) represents an alkyl group having the above concept unless otherwise specified. ],

Alkenyl group [This means an alkenyl group consisting of linear, branched, cyclic or a combination thereof, and the alkenyl group may be substituted or unsubstituted. A linear or branched, substituted or unsubstituted alkenyl group (preferably a linear or branched, substituted or unsubstituted alkenyl group having 2 to 30 carbon atoms, for example, vinyl group, allyl group, prenyl) Group, geranyl group, oleyl group, etc., a substituted or unsubstituted cycloalkenyl group (preferably a substituted or unsubstituted cycloalkenyl group having 3 to 30 carbon atoms, that is, hydrogen of a cycloalkene having 3 to 30 carbon atoms) A monovalent residue obtained by removing one atom, for example, a 2-cyclopenten-1-yl group, a 2-cyclohexen-1-yl group, etc., a substituted or unsubstituted bicycloalkenyl group (preferably having a carbon atom number). 5 to 3
0 is a substituted or unsubstituted bicycloalkenyl group, that is, a monovalent residue obtained by removing one hydrogen atom of a bicycloalkene having one or more double bonds, and is, for example, bicyclo [2,2,1] hept-2- En-1-yl group, bicyclo [2,2,2] oct-2-en-4-yl) group],
An alkynyl group (preferably a substituted or unsubstituted alkynyl group having 2 to 30 carbon atoms, for example, an ethynyl group, a propargyl group, a trimethylsilylethynyl group, etc.),

Aryl group (preferably substituted or unsubstituted aryl group having 6 to 30 carbon atoms, such as phenyl group, p-tolyl group, naphthyl group, m-chlorophenyl group, o-hexadecanoylaminophenyl group) A heterocyclic group (preferably a 5- or 6-membered substituted or unsubstituted heterocyclic group, that is, a 5- or 6-membered substituted or unsubstituted aromatic heterocyclic compound or a non-aromatic heterocyclic compound with a single hydrogen atom The removed monovalent residue is more preferably a 5- or 6-membered aromatic heterocyclic group having 3 to 30 carbon atoms, for example, 2-furyl group, 2-thienyl group, 2-pyrimidinyl group. , 2-benzothiazolyl group, aziridyl group, etc., cyano group, hydroxyl group, nitro group, carboxyl group, alkoxy group (preferably a group having 1 to 30 carbon atoms). Or an unsubstituted alkoxy group include a methoxy group, an ethoxy group, an isopropoxy group, t
-Butoxy group, n-octyloxy group, 2-methoxyethoxy group, etc.), aryloxy group (preferably substituted or unsubstituted aryloxy group having 6 to 30 carbon atoms, for example, phenoxy group, 2-methylphenoxy group) Base,
4-t-butylphenoxy group, 3-nitrophenoxy group, 2-tetradecanoylaminophenoxy group, etc.),
A silyloxy group (preferably a silyloxy group having 3 to 20 carbon atoms, for example, a trimethylsilyloxy group,
t-butyldimethylsilyloxy group, etc.),

A heterocyclic oxy group (preferably a substituted or unsubstituted heterocyclic oxy group having 2 to 30 carbon atoms, for example, 1-phenyltetrazole-5-oxy group, 2-
Tetrahydropyranyloxy group, etc., acyloxy group (preferably formyloxy group, C2 to C30)
A substituted or unsubstituted alkylcarbonyloxy group, a substituted or unsubstituted arylcarbonyloxy group having 6 to 30 carbon atoms, for example, a formyloxy group, an acetyloxy group, a pivaloyloxy group, a stearoyloxy group, a benzoyloxy group, p -Methoxyphenylcarbonyloxy group, etc.), carbamoyloxy group (preferably a substituted or unsubstituted carbamoyloxy group having 1 to 30 carbon atoms, for example, N, N-dimethylcarbamoyloxy group, N, N-diethylcarbamoyloxy group). Group, morpholinocarbonyloxy group, N, N-di-n-octylaminocarbonyloxy group, N-n-octylcarbamoyloxy group, etc., alkoxycarbonyloxy group (preferably substituted or unsubstituted with 2 to 30 carbon atoms). Substituted alkoxycarbonyl Shi group, for example methoxycarbonyloxy group, ethoxycarbonyloxy group, t-butoxycarbonyl group, such as n- octyl carbonyloxy group),

Aryloxycarbonyloxy group (preferably a substituted or unsubstituted aryloxycarbonyloxy group having 7 to 30 carbon atoms, for example, phenoxycarbonyloxy group, p-methoxyphenoxycarbonyloxy group, pn- Hexadecyloxyphenoxycarbonyloxy group, etc.), amino group (preferably unsubstituted amino group, substituted or unsubstituted alkylamino group having 1 to 30 carbon atoms, substituted or unsubstituted anilino group having 6 to 30 carbon atoms) Group, for example, amino group, methylamino group, dimethylamino group, anilino group, N-methyl-anilino group, diphenylamino group, etc., acylamino group (preferably formylamino group, having 1 to 3 carbon atoms)
0 substituted or unsubstituted alkylcarbonylamino group, substituted or unsubstituted arylcarbonylamino group having 6 to 30 carbon atoms, for example, formylamino group, acetylamino group, pivaloylamino group, lauroylamino group, benzoylamino group, 3,4,5-tri-n-octyloxyphenylcarbonylamino group, etc., aminocarbonylamino group (preferably a substituted or unsubstituted aminocarbonylamino group having 1 to 30 carbon atoms, for example, carbamoylamino group, N, N-dimethylaminocarbonylamino group, N, N-diethylaminocarbonylamino group, morpholinocarbonylamino group, etc.),

Alkoxycarbonylamino group (preferably a substituted or unsubstituted alkoxycarbonylamino group having 2 to 30 carbon atoms, for example, methoxycarbonylamino group, ethoxycarbonylamino group, t-butoxycarbonylamino group, n-octadecyloxy group. Carbonylamino group, N-methyl-methoxycarbonylamino group, etc., aryloxycarbonylamino group (preferably substituted or unsubstituted aryloxycarbonylamino group having 7 to 30 carbon atoms, for example, phenoxycarbonylamino group, p -Chlorophenoxycarbonylamino group, m-n-octyloxyphenoxycarbonylamino group, etc., sulfamoylamino group (preferably a substituted or unsubstituted sulfamoylamino group having 0 to 30 carbon atoms, for example, sulfone). Amoiruamino group, N, N-
Dimethylaminosulfonylamino group, N-n-octylaminosulfonylamino group, etc., alkylsulfonylamino group or arylsulfonylamino group (preferably substituted or unsubstituted alkylsulfonylamino group having 1 to 30 carbon atoms, carbon atom number) 6 to 30 substituted or unsubstituted arylsulfonylamino group, for example, methylsulfonylamino group, butylsulfonylamino group, phenylsulfonylamino group, 2,3,5-trichlorophenylsulfonylamino group, p-methylphenylsulfonylamino group Such),

A mercapto group, an alkylthio group (preferably a substituted or unsubstituted alkylthio group having 1 to 30 carbon atoms, for example, a methylthio group, an ethylthio group, an n-hexadecylthio group, etc.), an arylthio group (preferably having 6 carbon atoms). To 30 substituted or unsubstituted arylthio groups such as phenylthio group, p-chlorophenylthio group, m-methoxyphenylthio group and the like, heterocyclic thio group (preferably substituted or unsubstituted with 2 to 30 carbon atoms). Heterocyclic thio groups such as 2-benzothiazolylthio group, 1-phenyltetrazol-5-ylthio group, etc., sulfamoyl groups (preferably substituted or unsubstituted sulfamoyl groups having 0 to 30 carbon atoms, such as N
-Ethylsulfamoyl group, N- (3-dodecyloxypropyl group) sulfamoyl group, N, N-dimethylsulfamoyl group, N-acetylsulfamoyl group, N-benzoylsulfamoyl group, N- (N '-Phenylcarbamoyl) sulfamoyl group), an alkylsulfinyl group or an arylsulfinyl group (preferably a substituted or unsubstituted alkylsulfinyl group having 1 to 30 carbon atoms, a substituted or unsubstituted arylsulfinyl group having 6 to 30 carbon atoms, For example, methylsulfinyl group, ethylsulfinyl group, phenylsulfinyl group, p-methylphenylsulfinyl group, etc., alkylsulfonyl group or arylsulfonyl group (preferably a substituted or unsubstituted alkylsulfonyl group having 1 to 30 carbon atoms, 6 to 30 substitutions or no placement Substituted arylsulfonyl group, for example, methylsulfonyl group, ethylsulfonyl group, phenylsulfonyl group, p-methylphenylsulfonyl group, etc.),

Acyl group (preferably formyl group, substituted or unsubstituted alkylcarbonyl group having 2 to 30 carbon atoms, substituted or unsubstituted arylcarbonyl group having 7 to 30 carbon atoms, for example, acetyl group, pivaloyl group Two
-Chloroacetyl group, stearoyl group, benzoyl group,
pn-octyloxyphenylcarbonyl group, etc.),
Aryloxycarbonyl group (preferably a substituted or unsubstituted aryloxycarbonyl group having 7 to 30 carbon atoms, for example, phenoxycarbonyl group, o-chlorophenoxycarbonyl group, m-nitrophenoxycarbonyl group, pt-butyl Phenoxycarbonyl group etc.),
Alkoxycarbonyl group (preferably a substituted or unsubstituted alkoxycarbonyl group having 2 to 30 carbon atoms, for example, methoxycarbonyl group, ethoxycarbonyl group, t
-Butoxycarbonyl group, n-octadecyloxycarbonyl group and the like), carbamoyl group (preferably substituted or unsubstituted carbamoyl having 1 to 30 carbon atoms, for example, carbamoyl group, N-methylcarbamoyl group, N,
N-dimethylcarbamoyl group, N, N-di-n-octylcarbamoyl group, N- (methylsulfonyl) carbamoyl group, etc.),

An arylazo group or a heterocyclic azo group (preferably a substituted or unsubstituted arylazo group having 6 to 30 carbon atoms, a substituted or unsubstituted heterocyclic azo group having 3 to 30 carbon atoms, for example, a phenylazo group, p-chlorophenylazo group, 5-ethylthio-1,3,4-thiadiazol-2-ylazo group, etc.), imide group (preferably N-succinimide group, N-phthalimide group, N-
Maleimide group etc.), phosphino group (preferably substituted or unsubstituted phosphino group having 2 to 30 carbon atoms, for example, dimethylphosphino group, diphenylphosphino group, methylphenoxyphosphino group etc.), phosphinyl group (preferably Is a substituted or unsubstituted phosphinyl group having 2 to 30 carbon atoms, for example, phosphinyl group, dioctyloxyphosphinyl group, diethoxyphosphinyl group, etc., phosphinyloxy group (preferably having carbon atoms) 2 to 30 substituted or unsubstituted phosphinyloxy groups, for example, diphenoxyphosphinyloxy group, dioctyloxyphosphinyloxy group, etc., phosphinylamino group (preferably having 2 to 30 carbon atoms). A substituted or unsubstituted phosphinylamino group, such as dimethoxyphosphinylamido. Groups, such as dimethylamino phosphinyl amino group), a silyl group (preferably, having 3 carbon atoms
To 30 substituted or unsubstituted silyl groups, for example, trimethylsilyl group, t-butyldimethylsilyl group, phenyldimethylsilyl group, etc.), isothiocyanate group, and isocyanate group.

Preferred alkyl groups represented by R include, for example, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, n-pentyl group, n-hexyl group, n-heptyl group. , Unsubstituted alkyl groups such as n-octyl group, n-nonyl group and n-decyl group, sulfoalkyl group or a salt thereof, carboxyalkyl group or a salt thereof, hydroxyalkyl group, aminoalkyl group or a salt thereof, aryl Substituted alkyl groups such as alkyl groups can be mentioned. As the substituted alkyl group, more specifically, 5-ethoxycarbonylpentyl group, 3-ethoxycarbonylpropyl group, 3-aminopropyl group, 2- (2-aminoethoxy) ethyl group, 3-
Hydroxypropyl group, 2-methoxyethyl group, 2-hydroxyethyl group, 4-bromobutyl group, 2-cyanoethyl group, 3-nitrobenzyl group, 2-propen-1-yl group, allyl group, 2-sulfoethyl group, 3 -Sulfopropyl group, 4-sulfobutyl group, benzyl group and the like can be mentioned. The alkyl groups represented by R ¹ , R ² , R ³ and R ⁴ are the same as R above.

Y ¹ and Y ² are each independently a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, an alkynyl group having 2 to 6 carbon atoms, or an alkoxy group having 1 to 6 carbon atoms. (Methoxy group, ethoxy group, etc.), 6 to 6 carbon atoms
10 aryl groups (phenyl group, naphthyl group, etc., as well as aryl groups containing at least one hetero atom as a ring-constituting atom, for example, pyridinyl group, quinolyl group, thienyl group,
It includes a furyl group and an oxazolyl group. The same applies to other substituents containing an aryl moiety. ), Carbon number 6 to
10 aryloxy groups (phenoxy group, naphthyloxy group, pyridyloxy group, etc.), sulfo group, cyano group,
Halogen atom, carboxyl group, alkoxycarbonyl group having 1 to 6 carbon atoms (methoxycarbonyl group, ethoxycarbonyl group, etc.), carbamoyl group having 1 to 6 carbon atoms (unsubstituted carbamoyl group, monomethylcarbamoyl group, dimethylcarbamoyl group, etc.), Amino groups having 0 to 6 carbon atoms (unsubstituted amino group, methylamino group, dimethylamino group, ethylmethylamino group, etc.), sulfonyl groups having 0 to 10 carbon atoms (unsubstituted sulfonyl group, alkylsulfonyl group, etc.), carbon A number of 0 to 10 sulfamoyl group (unsubstituted sulfamoyl group, alkylsulfamoyl group, etc.),
Nitro group, carbonamido group having 1 to 6 carbon atoms, 1 to carbon number
6 sulfonamide group, ureido group having 1 to 6 carbon atoms, alkoxycarbonylamino group having 2 to 6 carbon atoms (methoxycarbonylamino group, ethoxycarbonylamino group, etc.), alkylthio group having 1 to 6 carbon atoms (methylthio group,
Ethylthio group, etc.), arylthio group having 6 to 10 carbon atoms (phenylthio group, naphthylthio group, etc.), 1 to 1 carbon atoms
6 is an aromatic heterocyclic thio group (the aromatic heterocycle may be any of those exemplified above. Examples of the aromatic heterocyclic thio group are pyridylthio group, imidazolylthio group, oxazolylthio group, etc.) or hydroxyl group . Y
These groups represented by ¹ and Y ² may have a substituent. As the substituent, those exemplified above can be used. Preferably, as Y ¹ and Y ² , a hydrogen atom, a sulfo group or a salt thereof, a halogen atom, a substituted or unsubstituted aryl group (eg, phenyl group), a substituted or unsubstituted alkynyl group (eg, substituted or unsubstituted phenyl). Examples thereof include an alkynyl group substituted with a group).

The compounds of the present invention represented by the above general formulas (I) to (IV) may have one or more asymmetric carbon atoms, and stereoisomers such as optically active substances or diastereoisomers based on the asymmetric carbon atoms. There may be isomers. Any stereoisomers in pure form, mixtures of any stereoisomers, racemates, etc. are all within the scope of the present invention. Further, the compound of the present invention may have an olefinic double bond, but the configuration may be either E or Z, and may be present as a mixture of both. Although the compounds of the present invention may exist as tautomers, any tautomers or mixtures thereof are included within the scope of the present invention. Furthermore, the above general formulas (I) to (IV)
The compound of the present invention represented by may form a salt depending on the kind of the substituent, and the free form compound or the salt form compound may form a hydrate or a solvate. All such substances are included within the scope of the present invention.

The type of salt is not particularly limited, but when it has a carboxyl group or a sulfo group, for example, a metal salt (sodium salt, potassium salt, calcium salt, magnesium salt), ammonium salt, or organic amine salt (triethylamine salt). , Dimethylamine salt, etc.). When a basic functional group is present, preferable anion species that form a counter ion include, for example, a halide ion (chloride ion, bromide ion, iodo ion, etc.), a sulfonate ion (methanesulfonate ion, paratoluene, etc.). Sulfonate ion, benzenesulfonate ion, etc.), perchlorate ion, sulfate ion, hydrogen sulfate ion, phosphate ion, hydrogen phosphate ion, sulfate monoester ion (monomethyl sulfate ion,
Monoethylsulfate ion, etc.) Boron tetrafluoride ion,
Examples thereof include hexafluorophosphate ion and carboxylate ion (acetate ion, propionate ion, oxalate ion, tartrate ion, etc.).

The compound of general formula (I) can be synthesized by the following synthetic route. Any synthetic reaction can be accomplished by using reactions known in the art.

[Chemical 5]

As a general synthetic method considered to give 2,3,3-trimethyl-3H-pyrrolo [2,3-b] pyridine-5-sulfonic acid represented by the general formula (I),
2,3,3-Trimethyl-3H-pyrrolo [2,3-b]
A method of heating pyridine in concentrated sulfuric acid is conceivable, but according to the study by the present inventors, the target product was not obtained no matter how the temperature or the concentration of the reagent was changed.
The compound obtained before and after was found to be (2,3-dimethyl-3H-pyrrolo [2,3-b] pyridin-3-yl) methylsulfonic acid. Therefore, depending on the method of directly sulfonating 2,3,3-trimethyl-3H-pyrrolo [2,3-b] pyridine, 2,3,3-of the general formula (I) is used.
Trimethyl-3H-pyrrolo [2,3-b] pyridine-5
Obtaining the sulfonic acid can be extremely difficult.

The compound represented by the general formula (II) can be easily synthesized from the compound represented by the general formula (I). As a general synthesis method, a compound represented by the general formula (I) and an alkylating reagent represented by R-Z (Z is a leaving group) are added at 40 ° C. in the presence or absence of a solvent. ~ 18
It can be produced by heating to about 0 ° C. When the compound represented by the general formula (I) has low solubility in the reaction system, M in the general formula (I) is converted into an organic cation to form R-Z.
The method of reacting with can also be preferably used. Z may combine with R in the molecule to form a cyclic structure. In this case, propane sultone, butane sultone, β-propyrolactone and the like can be used as the alkylating reagent. After reaction with the above alkylating reagent, the compound of general formula (I
It is possible to remove excess alkylating reagent, other solvents, raw material reagents, impurities, etc. by using a solvent having a low solubility of the target compound represented by I) such as ethyl acetate, toluene, diethyl ether, acetone and the like. it can. By carrying out the purification in this manner, the compound represented by the general formula (III) or (I
It is possible to obtain a compound represented by the general formula (II) having a sufficient purity for producing the fluorescent dye represented by V).

Specific examples of the compounds represented by formulas (I) and (II) are shown below, but the scope of the present invention is not limited to these examples.

[0025]

[Chemical 6]

[0026]

[Chemical 7]

[0027]

[Chemical 8]

The compound represented by the general formula (III) or (IV) of the present invention can be produced by using the compound of the above general formula (I) or (II). Since the compound represented by the general formula (II) is in equilibrium with a nucleophilic compound called methylene base due to heating or the presence of a base, the compound represented by the general formula (III) is reacted with an electrophile under various conditions. Or (I
It is possible to lead to a dye represented by V). Representative synthetic methods of the dyes represented by the general formulas (III) and (IV) of the present invention are shown in the following schemes (wherein the general formula (II
I) was taken as an example). For the production of the compounds represented by the general formulas (III) and (IV), the two routes shown in the scheme can be appropriately selected.

[0029]

[Chemical 9]

In the above scheme, the other 3H-pyrrolo [2, which binds to the quaternary salt represented by the general formula (II),
The compound having a 3-b] pyridine skeleton can be produced by applying the method shown as the synthesis method of the general formula (I) described above, but in Examples of the present specification, 5-halo-3H is used.
-Pyrrolo [2,3-b] pyridine skeleton and 5-aryl-
The synthesis of the 3H-pyrrolo [2,3-b] pyridine skeleton is specifically shown. 3H-pyrrolo [2,3-obtained here
The b] pyridine compound can be converted into a quaternary salt by the method described above as a method for converting the compound represented by the general formula (I) into the compound represented by the general formula (II).

Regarding the dye represented by the general formula (IV), the other 3H bound to the quaternary salt represented by the general formula (II)
Examples of compounds having a -pyrrolo [2,3-b] pyridine skeleton include 5-bromo-2,3,3- shown in Examples.
It can be produced by using a monoalkylhydrazine such as monomethylhydrazine or 3-sulfopropylhydrazine in place of hydrazine monohydrate in the method for synthesizing trimethyl-3H-pyrrolo [2,3-b] pyridine. At this time, the product may be obtained as a quaternary salt or may be obtained as a compound having an electrically neutral enamine structure due to dissociation of the proton on the methyl group bonded to the 2-position. It can be preferably used.

Specific examples of the compounds represented by the general formulas (III) and (IV) are shown below, but the scope of the present invention is not limited to these specific examples.

[0033]

[Chemical 10]

[0034]

[Chemical 11]

[0035]

[Chemical 12]

[0036]

[Chemical 13]

Regarding the method for producing a cyanine dye, for example, FM Hamer in The Cyanine Dyesand Related Comp
ounds, John Wiley and Sons, New York, 1964, Cytome
try, 11, pp.416-430, 1990; Cytometry, 12, pp.723-73
0, 1990; Bioconjugate Chem., 4, pp.105-111, 1993;
Anal. Biochem. 217, pp.187-204, 1994; Tetrahedron
45, pp. 4845-4866, 1989; European Patent Application 0591820.
It is also possible to refer to known methods for producing cyanine dye compounds described in, for example, A1 and European Patent Application 0580145A1. Since the examples of the present specification disclose specific methods for producing representative examples of the compounds of the present invention, those skilled in the art can refer to the methods described in the examples and, if necessary, It is possible to produce a dye compound included in the above general formula by appropriately modifying or modifying these methods and further selecting starting materials or reagents. In the synthesis, various condensation reactions, addition reactions, oxidation reactions, reduction reactions and the like can be combined in one step or a plurality of steps. These are detailed in the books. For example, various methods and raw material compounds described as unit operations in "Experimental Chemistry Course" (published by Maruzen Co., Ltd., each volume contained in each edition from the first edition to the fourth edition can be used) are preferably used. it can.

For example, in these production methods, when the defined group is changed under the conditions of the reaction step or is unsuitable for carrying out the reaction step, a method commonly used in synthetic organic chemistry, for example, In some cases, a desired step can be efficiently performed by applying a means such as protection or deprotection of a functional group or a treatment such as oxidation, reduction, hydrolysis or the like. The production intermediate and the target compound in the above steps are
Purification methods commonly used in synthetic organic chemistry, such as filtration, extraction,
It can be isolated and purified by treatments such as washing, drying, concentration, recrystallization and various chromatographies.
In addition, the production intermediate can be subjected to the next reaction without isolation.

[0039]

The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to the following examples. In the examples below, the compound numbers correspond to the compound numbers given above as preferred compounds. Example 1: 2,3,3-trimethyl-3H-pyrrolo [2,3
-B] Synthesis of pyridine-5-sulfonic acid sodium salt (1-1) Synthesis of 2-aminopyridine-5-sulfonic acid 104 g of 2-aminopyridine was dissolved in 280 g of concentrated sulfuric acid, and the pressure was reduced with an aspirator. 170 in an oil bath
The mixture was heated to ℃ and reacted for 4 hours. After cooling, 250 mL of methanol was added and cooled with ice water to precipitate crystals. The crystals were collected by filtration, washed with methanol, washed with ethyl acetate and then dried to obtain the desired product. Yield 128g,
Yield 66.5% nmr data (DMSO-d ₆ ) δ 3.45 (2
H, bs), 6.94 (1H, d), 8.0 (1H, d
d), 8.06 (1H, d), 8.15 (1H, bs)

(1-2) 2-hydroxypyridine-5-
Synthesis of Sulfonic Acid 120 g of 2-aminopyridine-5-sulfonic acid was added to 70
After dissolving in 0 mL of warm water and adding 100 g of sulfuric acid,
Cooled with ice water. 70.5 g of sodium nitrite solution (dissolved in 170 mL of water) was slowly added dropwise to this reaction solution. After completion of dropping, the mixture was stirred for 2 hours, then the solvent was distilled off and the residue was dried. The residue was heated to add a minimum amount of water in which the solid matter was dissolved, and cooled to precipitate crystals. The crystals were collected by filtration and dried to obtain the desired product. Yield 65 g, yield 53.
9% nmr data (DMSO-d ₆ ) δ 6.28 (1
H, d), 7.52 (1H, d), 7.58 (1H, d
d), 11.45 (1H, bs)

(1-3) Synthesis of 2-chloropyridine-5-sulfonic acid To 175 g of 2-hydroxypyridine-5-sulfonic acid, 50 mL of dimethylformamide was added and stirred. Heat to 100 ° C. on an oil bath and slowly add 480 g of thionyl chloride dropwise. After completion of the dropping, the reaction temperature was adjusted to 120 ° C., and 300 mL of thionyl chloride was further added. After completion of dropping, the reaction was carried out for 2 hours under the same conditions, and after cooling, the reaction solution was poured into ice. Ethyl acetate was added, extraction was performed twice, the organic phase was dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Then, concentrated oil (100 mL) and methanol (500 mL) were added to the concentrated oily material, the mixture was refluxed for 10 hours, and the reaction solution was concentrated under reduced pressure. Water was added to the residue to precipitate crystals, which were collected by filtration. Yield 60 g, yield 31.0% nmr data (DMSO-d ₆ ) δ 7.04 (1
H, d), 8.01 (1H, dd), 8.11 (1H,
d)

(1-4) 2-hydrazinopyridine-5-
Synthesis of sulfonic acid 20 g in 40 g of 2-chloropyridine-5-sulfonic acid.
7 g of hydrazine monohydrate was added and heated to 120 ° C. After reacting for 5 hours and cooling, crystals were precipitated.
This was collected by filtration to obtain the desired product. Yield 22.6g, yield 5
7.7% nmr data (DMSO-d ₆ ) δ 6.71 (1
H, d), 7.1 (4H, bs), 7.77 (1H, d
d), 8.20 (1H, d)

(1-5) 2,3,3-trimethyl-3H
-Synthesis of pyrrolo [2,3-b] pyridine-5-sulfonic acid sodium salt To 20 g of 2-hydrazinopyridine-5-sulfonic acid, 40 mL of water was added, and 4 g of sodium hydroxide was added. To this, 40 g of 3-methyl-2-butanone was added, and the mixture was heated under reflux for 1 hour, and then the solvent was distilled off under reduced pressure. 3 left
Add 0 mL of 1,4-butanediol and add 1 on an oil bath.
It heated at 90 degreeC and reacted for 4 hours. 10 g of sodium acetate was added to the reaction solution, and the mixture was directly subjected to silica gel column chromatography. After eluting with a mixed solvent of chloroform / methanol, the target substance was traced by developing TLC on silica gel and then paraanisaldehyde / ethanol / concentrated sulfuric acid =
The solution of 5/90/5 was sprayed and heated on a hot plate to collect and concentrate the eluate containing an orange-colored component. Dissolve this concentrate in methanol,
Purification was performed 3 times by column chromatography (methanol elution) using Sephadex LH-20 as a carrier, and further 1 column chromatography using alumina as a carrier.
The product was purified twice to obtain the desired product. Yield 0.6 g, yield 2.4% nmr data (DMSO-d ₆ ) δ 1.29 (6
H, s), 2.29 (3H, s), 7.99 (1H,
d), 8.53 (1H, d)

Example 2 Synthesis of (2-1) 2,3,3,7-tetramethyl-3H-pyrrolo [2,3-b] pyridinium-5-sulfonic acid inner salt 20 mg 2,3,3- Trimethyl-3H-pyrrolo [2,3-b] pyridine-5-sulfonic acid, 15 mg of triethylamine and 35 mg of methyl paratoluenesulfonate were mixed, heated to 140 ° C. and reacted for 40 minutes. After cooling, ethyl acetate was added, the components dissolved in ethyl acetate were removed by decantation, and this operation was repeated 3 times. The desired product was obtained as a residue. Yield 11 mg. MS spectrum (255, M + 1)

(2-2) 7-Ethyl-2,3,3-trimethyl-3H-pyrrolo [2,3-b] pyridinium-5
-Synthesis of inner salt of sulfonic acid Using ethyl paratoluenesulfonate in place of methyl paratoluenesulfonate used in Synthesis Example (2-1),
The same operation was performed to obtain the desired product. Yield 11 mg. MS spectrum (269, M + 1)

(2-3) 7-n-butyl-2,3,3-
Synthesis of trimethyl-3H-pyrrolo [2,3-b] pyridinium-5-sulfonic acid inner salt 20 mg 2,3,3-trimethyl-3H-pyrrolo [2,3-b] pyridine-5-sulfonic acid, 15 mg of triethylamine and 40 mg of butyl paratoluenesulfonate were mixed, heated to 150 ° C., and reacted for 1 hour. After cooling, ethyl acetate was added, the components dissolved in ethyl acetate were removed by decantation, and this operation was repeated 3 times. The desired product was obtained as a residue. Yield 10 mg. MS spectrum (383, M + 1)

(2-4) Synthesis of 7- (5- (ethoxycarbonyl) pentyl) -2,3,3-trimethyl-3H-pyrrolo [2,3-b] pyridinium-5-sulfonic acid inner salt 20 mg 2,3,3-trimethyl-3H-pyrrolo [2,3-b] pyridine-5-sulfonic acid, 15 mg triethylamine and 40 mg ethyl 6-bromohexanoate are mixed and heated to 150 ° C. for 45 minutes. Reacted After cooling, ethyl acetate was added, the components dissolved in ethyl acetate were removed by decantation, and this operation was repeated 3 times. The desired product was obtained as a residue. Yield 15 mg. MS spectrum (385, M + 1)

(2-5) Synthesis of 7- (3- (ethoxycarbonyl) propyl) -2,3,3-trimethyl-3H-pyrrolo [2,3-b] pyridinium-5-sulfonic acid inner salt 20 mg 2,3,3-trimethyl-3H-pyrrolo [2,3-b] pyridine-5-sulfonic acid, 15 mg triethylamine and 35 mg ethyl 4-bromobutyrate were mixed, heated to 140 ° C. and reacted for 30 minutes. did. After cooling, ethyl acetate was added, the components dissolved in ethyl acetate were removed by decantation, and this operation was repeated 3 times. The desired product was obtained as a residue. Yield 12 mg. MS spectrum (327, M + 1)

(2-6) 7- (3-Sulfopropyl)-
2,3,3-Trimethyl-3H-pyrrolo [2,3-b]
Synthesis of pyridinium-5-sulfonic acid inner salt 22 mg 2,3,3-trimethyl-3H-pyrrolo [2,3-b] pyridine-5-sulfonic acid sodium salt, 0.4 mL dimethylformamide and 40 mg.
Of propane sultone is mixed and heated to 100 ℃, 30
It reacted for a minute. After cooling, ethyl acetate was added, the components dissolved in ethyl acetate were removed by decantation, and this operation was repeated 3 times. The desired product was obtained as a residue. MS spectrum (328, M + 1)

(2-7) 7- (4-sulfobutyl)-
2,3,3-Trimethyl-3H-pyrrolo [2,3-b]
Synthesis of pyridinium-5-sulfonic acid inner salt The propanesultone of (2-6) above was changed to butanesultone of the same mass, and the same operation was performed to obtain the desired product.

Example 3: Synthesis of 5-bromo-2,3,3-trimethyl-3H-pyrrolo [2,3-b] pyridine (3-1) Synthesis of 5-bromo-2-hydrazinopyridine is readily available. 125 g of 2,5-dibromopyridine, 350 ml of ethanol and 250 ml of hydrated hydrazine were mixed, and the mixture was heated under reflux for 30 hours. Ethanol was distilled off under reduced pressure, water was added, and the precipitated crystals were collected by filtration.
After washing with water and drying, 77 g of 5-bromo-2-hydrazinopyridine was obtained. Yield 77.6%.

(3-2) Synthesis of 5-bromo-2,3,3-trimethyl-3H-pyrrolo [2,3-b] pyridine 5-bromo-2-hydrazinopyridine 76 g, 3-methyl-2- Mix 100 ml of butanone and add 100
The reaction was carried out at 30 ° C for 30 minutes. After distilling off excess 3-methyl-2-butanone, 100 ml of 1,4-butanediol was added and the mixture was heated to 240 ° C. and refluxed for 5 hours. The reaction solution was cooled and directly subjected to silica gel column chromatography to confirm each fraction by nmr. Fractions containing the desired product were collected, the solvent was distilled off, and the product was crystallized from hexane / ethyl acetate to obtain the desired product as a single brown crystal. Yield 8.5 grams, yield 8.8%.

Example 4: Synthesis of 5-phenyl-2,3,3-trimethyl-3H-pyrrolo [2,3-b] pyridine 5-Bromo-2,3,3-trimethyl-3H-pyrrolo [2,3] -B] Pyridine (0.5 g), phenylboronic acid (0.38 g) and dimethylformamide (7 mL) were mixed, and tetrakis (triphenylphosphine) palladium (0.072 g) and potassium carbonate (0.9) were added under a nitrogen stream.
g was added to this. The reaction mixture was stirred, heated to 100 ° C. and reacted for 4 hours. After completion of the reaction, the mixture was extracted with chloroform, and the concentrate was purified by silica gel column chromatography. Chloroform / methanol was used as an eluent, and the target product was eluted with chloroform / methanol = 4/1. Fractions containing the desired product were collected and concentrated to obtain the desired product as a pale yellow solid. Yield 0.45g, Yield 9
1%.

Example 5 (5-1) Synthesis of dye (III-1) 2,3,3,7-tetramethyl-3H-pyrrolo [2,3-b] pyridinium synthesized in Synthesis Example (2-1) -5
10 mg of sulfonic acid inner salt and 30 mg of 1,3-diphenylformamidine were mixed, 1 mL of acetic anhydride was added, and a catalytic amount of p-toluenesulfonic acid monohydrate was added, and the mixture was heated to 60 ° C. Reacted for 20 minutes. The reaction mixture is cooled to room temperature, 1 mL of pyridine is added,
After adding g of 2,3,3,7-tetramethyl-3H-pyrrolo [2,3-b] pyridinium-5-sulfonic acid inner salt, 0.1 mL of triethylamine was added dropwise. Since the target dye is formed, it is subjected to silica gel column chromatography and Sephadex L.
Purification was carried out using an H-20 / methanol elution system to obtain the desired product. MS spectrum (563, M + 2Na) λmax (methanol) 621 nm

(5-2) Synthesis of dye (III-4) 2,3,3-trimethyl-3H, which is a known compound in the literature
20 mg of pyrrolo [2,3-b] pyridine and 50 mg of propanesultone were mixed, heated to 100 ° C. and reacted for 30 minutes. After cooling, ethyl acetate was added to remove the components dissolved in ethyl acetate, then 40 mg of 1,3-diphenylformamidine was added, 2 mL of acetic anhydride and a catalytic amount of p-toluenesulfonic acid monohydrate were added. Then 60
The mixture was heated to ℃ and reacted for 20 minutes. After cooling to room temperature, 1 mL of pyridine was added, and 7- (5- (ethoxycarbonyl) pentyl) -2, synthesized by the method of Synthesis Example (2-4),
15 mg of 3,3-trimethyl-3H-pyrrolo [2,3-b] pyridinium-5-sulfonic acid inner salt was added,
Further, 0.1 mL of triethylamine was added. The ethyl ester form of the desired dye was produced. This was purified by silica gel column chromatography / chloroform-methanol elution, and Sephadex LH-20 / methanol elution system. Dissolve this dye in methanol,
The ester was hydrolyzed by adding 1 mol / L sodium hydroxide. After completion of the hydrolysis reaction, acetic acid was added to neutralize and after concentration, silica gel column chromatography /
Purification was performed using chloroform-methanol elution and Sephadex LH-20 / methanol elution system to obtain the desired product. MS spectrum (692, M + 2Na) λmax (methanol) 624 nm

(5-3) Synthesis of Dye (III-7) 5-Bromo-2,3,3-synthesized in Synthesis Example (3-2)
Trimethyl-3H-pyrrolo [2,3-b] pyridine 20
mg was mixed with 35 mg of methyl p-toluenesulfonate and heated at 130 ° C. for 1 hour. After cooling, ethyl acetate was added to remove components soluble in ethyl acetate, and then 40
1 mg of 1,3-diphenylformamidine, 2 mg of acetic anhydride and a catalytic amount of p-toluenesulfonic acid monohydrate were added, and the mixture was reacted at 70 ° C. for 1 hour. This reaction solution was cooled to room temperature and then synthesized by the method of Synthesis Example (2-4).
(5- (ethoxycarbonyl) pentyl) -2,3,3
12 mg of -trimethyl-3H-pyrrolo [2,3-b] pyridinium-5-sulfonic acid inner salt was added, and further 1 mL of pyridine and 0.1 mL of triethylamine were added. The ethyl ester form of the desired dye was produced. This is silica gel column chromatography / chloroform-
Purification was performed by elution with methanol. This dye was dissolved in methanol, and 1 mol / L sodium hydroxide was added to hydrolyze the ester portion. After completion of the hydrolysis reaction, acetic acid was added to neutralize and after concentration, purification was performed by silica gel column chromatography / chloroform-methanol elution and Sephadex LH-20 / methanol elution system to obtain the desired product. MS spectrum (618, M + 1) λmax (methanol) 622 nm

(5-4) Synthesis of dye (IV-2) 154.2 g of 3-sulfopropylhydrazine was added to 500
It was added to mL of ethylene glycol, 40 g of sodium hydroxide was added, and the mixture was heated and stirred to be dissolved. 150 g of 2-chloropyridine was added to this reaction solution, and the reaction was carried out at 150 ° C. until the hydrazine spot disappeared by TLC. After the reaction is complete, it is cooled and ethyl acetate is added,
The components dissolved in ethyl acetate were separated and discarded. Then, 120 g of 3-methyl-2-butanone was added to the solution of ethylene glycol, and the reaction was performed at 100 ° C. for 1 hour.
Hydrazone. The reaction solution was depressurized to distill off excess 3-methyl-2-butanone, then the temperature was raised to 190 ° C. and heating was continued for 4 hours. After cooling, ethylene glycol was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography to give 3- (1,2-dihydro-3,
3-Dimethyl-2-methylene-3H-pyrrolo [2,3-
b] Pyridin-1-yl) propanesulfonic acid sodium salt was obtained. The yield was 82 g and the yield was 26.9%.

To 100 mg of this compound, 300 mg of 1,3-diphenylformamidine was added, and 5% of acetic anhydride was added.
After adding mL and carrying out a reaction at 60 ° C. for 30 minutes, the temperature was brought to room temperature, and 7- (5- (ethoxycarbonyl) pentyl) -2,3,3-trimethyl-3 synthesized in Synthesis Example (2-4).
120 mg of H-pyrrolo [2,3-b] pyridinium-5-sulfonic acid inner salt was added. 2m pyridine
L and 0.3 mL of triethylamine were added, and the produced dye was produced by silica gel column chromatography to obtain an ethyl ester of the objective dye. This ester was dissolved in methanol, and 1 mol / L sodium hydroxide was added little by little until the reaction was completed. After completion of the reaction, acetic acid was added to neutralize and then concentrated, and purification was repeated by silica gel chromatography / chloroform-methanol elution system and Sephadex LH-20 / methanol elution system to obtain the desired product. MS spectrum (692, M + 2Na) λmax (methanol) 572 nm

(5-5) Synthesis of Dye (IV-3) 10 g of 2-chloropyridine-5-sulfonic acid obtained in Synthesis Example (1-3), 10 g of monomethylhydrazine and 20 mL of n-butanol were mixed and mixed. The mixture was heated under reflux for an hour. The solvent was distilled off, and concentrated hydrochloric acid was added to precipitate crystals. The crystals were collected by filtration, and 20 mL of 3-methyl-2-butanone, 2
After addition of 0 mL of ethylene glycol and heating reaction at 100 ° C. for 1 hour, excess 3-methyl-2-butanone was distilled off. Then, the reaction temperature was set to 180 ° C., the reaction was carried out for 5 hours, and after cooling, ethylene glycol was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography to obtain 1,2-dihydro-1,3,3-trimethyl. 2-Methylene-3H-pyrrolo [2,3-b] pyridine-5-sulfonic acid sodium salt was obtained.

To 10 mg of this compound, 30 mg of 1,3-diphenylformamidine was added, and further 1 mL of acetic anhydride was added.
Was added and reacted at 60 ° C. for 30 minutes, then brought to room temperature,
7- (5- (ethoxycarbonyl) pentyl) -2,3,3-trimethyl-3H- synthesized in Synthesis Example (2-4)
10 mg of pyrrolo [2,3-b] pyridinium-5-sulfonic acid inner salt was added. Further, 1 mL of pyridine and 0.1 mL of triethylamine were added, and the produced dye was produced by silica gel column chromatography to obtain an ethyl ester of the desired dye. This ester was dissolved in methanol, and 1 mol / L sodium hydroxide was added little by little until the reaction was completed. After completion of the reaction, acetic acid was added to neutralize and then concentrated, and purification was repeated by silica gel chromatography / chloroform-methanol elution system and Sephadex LH-20 / methanol elution system to obtain the desired product. MS spectrum (664, M + 2Na) λmax (methanol) 570 nm

(5-6) Synthesis of dye (IV-8) 3- (1,2-dihydro-) synthesized in Synthesis Example (5-4)
3,3-Dimethyl-2-methylene-3H-pyrrolo [2,2
3-b] pyridin-1-yl) propanesulfonic acid sodium salt 20 mg to malonaldehyde dianilide 80 m
g and acetic anhydride 2 mL were added, and the mixture was reacted at 60 ° C. for 1 hour. 7- (5-) synthesized in Synthesis Example (2-4) at room temperature
(Ethoxycarbonyl) pentyl) -2,3,3-trimethyl-3H-pyrrolo [2,3-b] pyridinium-5
18 mg of sulfonic acid inner salt was added. Further, 1 mL of pyridine and 0.1 mL of triethylamine were added, and the produced dye was produced by silica gel column chromatography to obtain an ethyl ester of the desired dye. This ester was dissolved in methanol, and 1 mol / L sodium hydroxide was added little by little until the reaction was completed. After completion of the reaction, acetic acid was added to neutralize and then concentrated, and purification was repeated by silica gel chromatography / chloroform-methanol elution system and Sephadex LH-20 / methanol elution system to obtain the desired product. MS spectrum (718, M + 2Na) λmax (methanol) 659 nm

The dyes of the present invention were compared for fluorescence and water solubility. The Cy3 and Cy5 carboxylic acid types marketed by Amersham Pharmacia shown below and dyes having the same skeleton as the present invention but having no sulfo group were compared. The water solubility is represented by 1 mM, which is a dye concentration generally used in the labeling reaction, as a standard, and when the solubility is more than 1 mM, it is represented as X. The maximum excitation wavelength and fluorescence intensity were measured as a methanol solution such that the absorbance at the maximum absorption wavelength was 0.1 and measured using a fluorometer. The fluorescence intensity was the fluorescence when excited at the maximum excitation wavelength. It represents the intensity of the photometer. From the results shown in Table 1 below, it can be seen that the fluorescent dye of the present invention has high fluorescence characteristics and is also excellent in water solubility.

[0063]

[Table 1]

[0064]

[Chemical 14]

[0065]

EFFECT OF THE INVENTION General formula (I) or general formula (II) of the present invention
The compound represented by is useful as an intermediate for producing an azacyanine dye. Further, the azacyanine dye represented by the general formula (III) or (IV) of the present invention has high fluorescence intensity and water solubility, and thus is useful as a marker for fluorescence labeling in the fields of biochemistry and gene diagnosis. is there.

   ─────────────────────────────────────────────────── ─── Continued front page    F term (reference) 4C065 AA04 BB04 CC01 DD02 EE02                       HH01 JJ09 KK02 LL01 PP01                       PP08                 4C072 MM02 UU05                 4H056 CA01 CC02 CC08 CE03 DD03                       FA08

Claims (2)

[Claims] 1. The following general formula (I) or general formula (II): (In the general formula (I), M represents a hydrogen atom or a counter cation of a sulfonate anion;
Represents a substituted or unsubstituted alkyl group having 10 or less carbon atoms) or a salt thereof. 2. The following general formula (III) or (IV): (In the formula, R ¹ , R ² , R ³ , and R ⁴ each independently represent a substituted or unsubstituted alkyl group having 10 or less carbon atoms; Y ¹
And Y ² are each independently a hydrogen atom, a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, a substituted or unsubstituted alkenyl group having 2 to 6 carbon atoms, a substituted or unsubstituted alkynyl group having 2 to 6 carbon atoms. Group, substituted or unsubstituted alkoxy group having 1 to 6 carbon atoms, substituted or unsubstituted aryl group having 6 to 10 carbon atoms, substituted or unsubstituted aryloxy group having 6 to 10 carbon atoms, sulfo group, cyano group A halogen atom, a carboxyl group, a substituted or unsubstituted alkoxycarbonyl group having 1 to 6 carbon atoms, a substituted or unsubstituted carbamoyl group having 1 to 6 carbon atoms, a substituted or unsubstituted amino group having 0 to 6 carbon atoms, A substituted or unsubstituted sulfonyl group having 0 to 10 carbon atoms, a substituted or unsubstituted sulfamoyl group having 0 to 10 carbon atoms, a nitro group, and 1 carbon atom
~ 6 substituted or unsubstituted carboxamide group, carbon number 1
~ 6 substituted or unsubstituted sulfonamide group, 1 to 6 carbon substituted or unsubstituted ureido group, 2 to 2 carbon atoms
6-substituted or unsubstituted alkoxycarbonylamino group, 1- to 6-carbon substituted or unsubstituted alkylthio group, 6- to 10-carbon substituted or unsubstituted arylthio group, 1- to 6-carbon substituted or unsubstituted An aromatic heterocyclic thio group or a hydroxyl group, n is 0 or 1)
Or a salt thereof.