HK1079110B - Near infrared fluorescent contrast agent and method for fluorescence imaging - Google Patents

Description

Near-infrared fluorescent contrast agent and method for fluorescence imaging

Technical Field

The present invention relates to a near-infrared fluorescent contrast agent, and a method for fluorescence imaging using the same.

Background

In treating diseases, it is important to detect morphological and functional changes caused by the disease in vivo at an early stage of the disease. Especially for cancer treatment, knowing the location and size of the tumor in advance is of paramount importance for determining further treatment strategies and protocols. Currently applied methods include biopsy by needle stick or the like and imaging diagnosis such as X-ray imaging, MRI, ultrasound imaging, and the like. Biopsy is an effective tool for definitive diagnosis, but imposes a significant burden on the patient to be diagnosed, and is also not suitable for tracking changes in the lesion over time. X-ray imaging and MRI inevitably expose the patient undergoing diagnosis to radiation or electromagnetic waves. In addition, the conventional imaging diagnosis as described above requires complicated operations and a long time in performing measurement and diagnosis. Large instruments also make it difficult to apply these methods during surgery.

One of the reported diagnostic imaging methods includes fluorescence imaging (lippspn r.l. et al, j.natl.cancer inst., 26, 1-11 (1961)). The method uses a substance as a contrast agent that emits fluorescence when exposed to excitation light of a particular wavelength. The method comprises the following steps: the body is exposed to excitation light outside the body and then the fluorescence emitted by the fluorescent contrast agent in the body is detected.

Examples of fluorescent contrast agents include, for example, porphyrin compounds, which accumulate in tumors and are used for photodynamic therapy (PDT), such as hematoporphyrin. Other examples include photopyrin and benzoporphyrins (see Lippspn R.L. et al, supra; Meng T.S. et al, SPIE, 1641, 90-98(1992), W084/04665, etc.). However, these compounds are phototoxic because they are originally intended for PDT (PDT requires these properties) and are therefore not ideal as diagnostic agents.

The use of known fluorescent dyes such as fluorescein, fluoroescamine and riboflavin for retinal circulatory microangiography is well known (U.S. 4945239). However, the fluorescence emitted by these fluorescent dyes in the visible region of 400-600nm only enables low transmission of living tissue and thus it is almost impossible to detect lesions in deeper parts of the body.

Cyanine compounds including indocyanine (indocyanine) green (hereinafter abbreviated as "ICG") are useful for the detection of liver function and cardiac output, and have been reported to be also useful as fluorescent contrast agents (haglund m. et al, Neurosurgery, 35, 930(1994), Li, x. et al, SPIE, 2389, 789-. The cyanine compound has absorption in the near-infrared region (700-1300 nm).

Near-infrared light has a high transmission property through living tissue and can pass through a skull of about 10cm, and for these reasons, attention has recently been paid to the light in the field of clinical medicine. For example, optical CT technology (CT technology using light transmission action of a medium) has been noted as a new technology in the clinical field because near-infrared light is permeable to a living body and oxygen concentration and circulation in the body can be detected using light in the range of the region.

Cyanine compounds emit fluorescence in the near infrared region, where light has excellent permeability in living tissues as described above, and thus have been proposed as fluorescent contrast agents. Various cyanine compounds have been developed in recent years, and have been used as fluorescent contrast agents (W096/17628, WP97/13490, etc.). However, no agent having a satisfactory ability to discriminate between damaged and normal tissues, i.e., an agent having a satisfactory selectivity for a site to be imaged, has been obtained yet.

Disclosure of Invention

The object of the present invention is to provide a fluorescent contrast agent that emits fluorescence in the near infrared region, has excellent permeability in living tissue, and enables tumor and/or blood vessel specific imaging. It is another object of the present invention to provide a method for fluorescence imaging using the near-infrared fluorescent contrast agent.

The present inventors have made various studies to achieve the above object. As a result, it was found that a fluorescent contrast agent having high tumor selectivity can be successfully obtained by introducing a carboxylic acid or an aryl group into a cyanine dye. They also successfully established a method for fluorescence imaging using the contrast agent. The present invention has been completed based on these findings.

Accordingly, the present invention provides a near-infrared fluorescent contrast agent comprising a compound represented by the following formula [ I ] or a pharmaceutically acceptable salt thereof:

wherein:

R¹、R²、R⁷and R⁸Independently of one another, represents substituted or unsubstituted C_1-10Alkyl or substituted or unsubstituted aryl, and R¹And R²And/or R⁷And R⁸Can be connected together to form a ring;

R³、R⁵、R⁵、R⁶、R⁹、R¹⁰、R¹¹and R¹²Independently of one another, represents a hydrogen atom, a substituted or unsubstituted C_1-6Alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, halogen, cyano, carboxyl or sulfo, and R³、R⁴、R⁵、R⁶、R⁹、R¹⁰、R¹¹And R¹²Can be connected together to form a ring;

X¹and X²Independently represents substituted or unsubstituted C_1-15Alkyl or substituted or unsubstituted aryl, and X¹And X²Having a total of 0-4 carboxyl groups, with the proviso that: when the number of carboxyl groups is 0 or 1, X¹And X²Each of which is C_1-5Carboxyalkyl or sulfoalkyl, and R³、R⁴、R⁵、R⁶、R⁹、R¹⁰、R¹¹And R¹²At least one of them represents a substituted or unsubstituted aryl group or a substituted or unsubstituted heteroaryl group;

m¹represents 0 or 1;

m²represents 0 or 1;

m³represents 0 or 1;

L¹、L²、L³、L⁴、L⁵、L⁶and L⁷Independently represents a substituted or unsubstituted methine group, with the proviso that: when two or more methines have a substituent, these substituents may be bonded to each other to form a ring, and when X has a substituent¹And X²When each has a carboxyl group, X¹And X²Are all carboxyl-substituted hydrocarbon radicals, and L¹、L²、L³、L⁴、L⁵、L⁶And L⁷At least one of the methines represented is a substituted methine group, and R⁴And R¹⁰Represents a sulfo group;

m represents a hydrogen atom, a metal or a quaternary ammonium salt; and

n represents an integer of 1 to 7 required for neutralizing the charge.

According to a preferred embodiment of the invention, m¹、m²And m³Are all simultaneously 1, and X¹Is a group represented by the following formula (i):

wherein Y is₁And Y₂Independently represents a substituted or unsubstituted divalent linking group.

According to a more preferred embodiment of the invention, X¹And X²Independently represents a group represented by the following formula (i):

wherein Y is₁And Y₂Independently represents a substituted or unsubstituted divalent bond.

According to a further preferred embodiment, R³、R⁴、R⁵、R⁶、R⁹、R¹⁰、R¹¹And R¹²At least one of which is a substituted or unsubstituted aryl group or a substituted or unsubstituted heteroaryl group. According to a still further preferred embodiment, R⁴、R⁵、R¹⁰And R¹¹Is a substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl; and X¹And X²Each is independently C_1-5Carboxyalkyl or sulfoalkyl.

According to another preferred embodiment of the present invention, X¹And X²Independently represents a group of the formula:

wherein Y is³Represents C_1-10A hydrocarbon radical, and L¹、L²、L³、L⁴、L⁵、L⁶And L⁷At least one of the methines represented is a substituted methine group, and R⁴And R¹⁰Represents a sulfo group.

Preferably, the number of sulfo groups in the molecule is 2 or less.

According to a further preferred embodiment, Y₁Represents- (CH)₂)_pCONH-, wherein p represents an integer of 1 to 4 and Y₂Represents- (CH)₂) -or (CH)₂)₂-。

The near-infrared fluorescent contrast agent can be preferably used for tumor imaging and angiography.

In another aspect, the present invention provides a method of performing fluorescence imaging, comprising the steps of: the near-infrared fluorescent contrast agent is introduced into a living body, the living body is exposed to excitation light, and then near-infrared fluorescence emitted from the contrast agent is detected.

Drawings

Fig. 1 is a graph showing the results of fluorescence imaging at a given time after administration of compound 2 of the present invention.

Fig. 2 is a graph showing the results of fluorescence imaging at a given time after ICG as a reference compound was administered.

Fig. 3 is a graph showing the results of fluorescence imaging at a given time after administration of compound a as a reference compound.

FIG. 4 is a schematic diagram of an experimental setup for fluorescence imaging performed in test example 2. In the figure, SHG represents second harmonic generation, THG represents third harmonic generation, and OPO represents an optical parametric oscillator.

Fig. 5 is a graph showing the results of fluorescence imaging at a given time after administration of compound 5 of the present invention.

Fig. 6 is a graph showing the results of fluorescence imaging at a given time after administration of compound 7 of the present invention.

Fig. 7 is a graph showing the results of fluorescence imaging at a given time after administration of compound 10 of the present invention.

Fig. 8 is a graph showing the results of fluorescence imaging at a given time after administration of compound B as a reference compound.

Detailed Description

R¹、R²、R⁷And R⁸C represents_1-10The alkyl group can be linear, branched, cyclic, or a combination thereof (the alkyl moiety in the alkyl group and the functional group comprising the alkyl group have the meanings as described in the specification unless otherwise specified). As unsubstituted alkyl groups, for example, methyl, ethyl, propyl, butyl and hexyl groups can be used. The number, kind or position of the substituent on the substituted alkyl group is not particularly limited. Examples of the substituted alkyl group include a sulfoalkyl group, a carboxyalkyl group, a hydroxyalkyl group, an alkoxyalkyl group, an aminoalkyl group, a haloalkyl group, a cyanoalkyl group, an aryl-substituted alkyl group, and a heteroaryl-substituted alkyl group.

R¹、R²、R⁷And R⁸The aryl radicals represented may be monocyclic or fused, and for example C may be used_6-14Aryl, preferably C_6-10Aryl (aryl and the aryl moiety in the functional group comprising aryl have the meaning as indicated in the specification unless otherwise specified). As aryl, phenyl or naphthyl can be preferably used, and phenyl is more preferred. As the substituted aryl group, a sulfophenyl group, a hydroxyphenyl group, or an aminophenyl group can be used.

In addition, R¹And R²、R⁷And R⁸Representative may be joined together to form a ring. Examples of the ring formed include, for example, a cyclopentyl ring, a cyclohexyl ring and the like. R¹、R²、R⁷And R⁸Preferably methyl or ethyl, more preferably methyl.

R³、R⁴、R⁵、R⁶、R⁹、R¹⁰、R¹¹And R¹²Each otherIndependently represents a hydrogen atom, a substituted or unsubstituted C_1-6Alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, halogen, cyano, carboxy or sulfo, and is selected from R³、R⁴、R⁵And R⁶And is selected from R⁹、R¹⁰、R¹¹And R¹²Two adjacent groups in (a) may independently be linked together to form a ring. The ring formed may be saturated or unsaturated, and may be a hydrocarbon ring or a heterocyclic ring. For example, R³And R⁴、R⁴And R⁵、R⁵And R⁶、R⁹And R¹⁰、R¹⁰And R¹¹Or R¹¹And R¹²May be linked together to form a benzene ring or an aromatic heterocyclic ring such as a pyridine ring. Preferred examples include R³And R⁴Or R⁹And R¹⁰Benzene rings formed by connecting them together.

As R³、R⁴、R⁵、R⁶、R⁹、R¹⁰、R¹¹And R¹²As the aryl group, for example, a phenyl group or a naphthyl group can be used. As heteroaryl, for example, thienyl, benzothienyl, furyl, benzofuryl, pyrrolyl, imidazolyl or quinolyl can be used. From 1 to 4 optional substituents may be present on the aryl and heteroaryl groups. The position of the substituent is not particularly limited, and when 2 or more substituents are present, they may be the same or different. For such substituents, for example, a hydroxyl group, a halogen atom such as fluorine, chlorine, bromine and iodine atoms; c_1-6Alkyl groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl; c_1-6Haloalkyl such as trifluoromethyl; c_1-6Alkoxy groups such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy; c_1-6Alkylenedioxy such as methylenedioxy, ethylenedioxy; a carboxyl group; c_1-6An alkoxycarbonyl group; an unsubstituted amino group; c_1-6Alkyl-substituted amino groups such as methylamino, dimethylamino, ethylamino; sulfo group(ii) a Or cyano, and the like.

X¹And X²Independently of one another, represents substituted or unsubstituted C_1-15Alkyl or substituted or unsubstituted aryl, and X¹And X²At X¹And X²Having a total of 1-4 carboxyl groups. As X¹And X²As the unsubstituted alkyl group, for example, a methyl group, an ethyl group, a propyl group, a butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, a pentyl group, an isopentyl group, a neopentyl group, a tert-pentyl group, a 2-methylpropyl group or a1, 1-dimethylpropyl group can be used. The alkyl group may be linear, branched, cyclic or a combination thereof, with linear or branched alkyl groups being preferred.

As X¹And X²As the substituted alkyl group, for example, sulfoalkyl groups (e.g., 2-sulfoethyl group, 3-sulfopropyl group, 3-methyl-3-sulfopropyl group, 4-sulfobutyl group and the like), carboxyalkyl groups (e.g., 1-carboxymethyl group, 2-carboxyethyl group, 3-carboxypropyl group, 4-carboxybutyl group and the like), hydroxyalkyl groups, alkoxyalkyl groups, aminoalkyl groups, haloalkyl groups, cyanoalkyl groups, heteroaryl-substituted alkyl groups, aryl groups or heteroaryl groups can be used. The alkyl portion of these groups has the same definition as the unsubstituted alkyl group defined above. As R¹、R²、R⁷And R⁸As the substituted or unsubstituted aryl group represented, a phenyl group, a sulfophenyl group, a hydroxyphenyl group or an aminophenyl group may be used.

When X is present¹And X²When the number of carboxyl groups in (1) is 0 or 1, C may be used_1-5Carboxyalkyl or sulfoalkyl as X¹And X²。

As Y₁And Y₂The divalent linking group represented by, for example, C which may be substituted or unsubstituted_1-6Alkylene groups such as methylene, ethylene, n-butylene, methylpropylene or phenylene. As another example, a linking group represented by the following formula may be used:

wherein q represents an integer from 1 to 4 and the symbol "·" represents a linking site. These hydrocarbon groups may have a substituent and may contain one or more hetero atoms. For example, they may contain ether, thioether, disulfide, amide, ester, sulfonamide, or sulfoester linkages.

As Y₁And Y₂The divalent linking group represented by, for example, a bond represented by the following formula may also be used:

wherein p represents an integer from 1 to 4 and the symbol "·" represents a linking site. Y is₁Preferred examples of (b) include a linking group represented by the following formula:

wherein p represents an integer of 1 to 4. Y is₁Most preferred is- (CH)₂)_p-CO-NH- (wherein p represents an integer from 1 to 4). Y is₂Preferred examples of (b) include methylene or ethylene.

L¹、L²、L³、L⁴、L⁵、L⁶And L⁷Independently represents a substituted or unsubstituted methine group, wherein m¹、m²And m³Independently represents 0 or 1. Preferably, m is¹、m²And m³Each simultaneously being 1. Examples of the substituent on the methine group include a substituted or unsubstituted alkyl group, a halogen atom, a substituted or unsubstituted aryl group, a lower alkoxy group or the like. Specific examples of substituted aryl groups include 4-chlorophenyl and the like. Lower alkoxy is preferably C_1-6Alkoxy, which may be linear or branched. Specific examples include methoxy, ethoxy, propoxy, butoxy, t-butoxy, pentyloxy, and the like, and methoxy or ethoxy is preferable. As substituents for methine groupsPreferably, methyl or phenyl is used.

When selected from L¹、L²、L³、L⁴、L⁵、L⁶And L⁷When the methine group in (1) is substituted, the substituents on the methine group may be bonded to each other to form a ring. Preferably, the substituents on the methine group may be linked to form a group comprising three consecutive substituents selected from L¹、L²、L³、L⁴、L⁵、L⁶And L⁷Ring of methine group(s) in (1). As in which the substituents on the methine group are linked to form a radical containing three consecutive substituents selected from L¹、L²、L³、L⁴、L⁵、L⁶And L⁷Examples of the ring of the methine group in (1) include, for example, those in which 4, 4-dimethylcyclohexene is formed so as to contain a ring L³、L⁴And L⁵The compound of (1). Wherein is selected from L¹、L²、L³、L⁴、L⁵、L⁶And L⁷Particularly preferred examples of the partial structure of the methine group forming a conjugated methine chain include a group represented by the following general formula (a):

wherein Z represents a non-metallic atom group necessary for forming a 5-or 6-membered ring, and A represents a hydrogen atom or a monovalent group.

Examples of the non-metallic atom group necessary for forming a 5-to 10-membered ring represented by Z include, for example, a carbon atom, a nitrogen atom, an oxygen atom, a hydrogen atom, a sulfur atom, a halogen atom (a fluorine atom, a chlorine atom, a bromine atom, an iodine atom) and the like. Examples of the 5-or 6-membered ring in the partial structure represented by the general formula (a) include, for example, a cyclopentene ring, a cyclohexene ring and a 4, 4-dimethylhexene ring, with a cyclopentene ring or a cyclohexene ring being preferred.

Examples of the monovalent group represented by A include, for example, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aralkyl group, a substituted or unsubstituted lower alkoxy group, a substituted or unsubstituted amino group, a substituted or unsubstituted alkylcarbonyloxy group (e.g., acetoxy group), a substituted or unsubstituted alkylthio group, a substituted or unsubstituted arylthio group, a cyano group, a nitro group, a halogen atom and the like.

Specific examples of the aralkyl group represented by A include benzyl, 2-phenylethyl, 3-phenylpropyl and the like. Examples of the substituent on the aralkyl group include, for example, a sulfo group, a carboxyl group, a hydroxyl group, a substituted or unsubstituted alkyl group, an alkoxy group, a halogen atom and the like. Specific examples of the substituted amino group represented by A include, for example, an alkylamino group (e.g., methylamino, ethylamino, etc.), a dialkylamino group (e.g., dimethylamino, diethylamino, etc.), a phenylamino group, a diphenylamino group, a methylphenylamino group, a cyclic amino group (e.g., morpholinyl, imidazolidinyl, ethoxycarbonylpiperadino, etc.), etc. If the substituted amino group has an additional substituent, a sulfo group, a carboxyl group, or the like may be used as the substituent. Specific examples of the arylthio group represented by A include a phenylthio group, a naphthylthio group and the like, and examples of the substituent of the arylthio group include a sulfo group, a carboxyl group and the like.

Examples of the monovalent group represented by A include phenylamino, diphenylamino, ethoxycarbonylpiperazinyl, arylthio and the like.

Y represents a non-metal atom necessary for forming a 5-to 10-membered heterocyclic ring, preferably a 5-to 6-membered heterocyclic ring (the heterocyclic ring may be a fused ring). Examples of the 5-to 10-membered heterocyclic ring formed by Y include the following rings: thiazole rings (e.g., thiazole, 4-methylthiazole, etc.), benzothiazole rings (e.g., benzothiazole, 4-chlorobenzothiazole, etc.), naphthothiazole rings (e.g., naphtho [2, 1-d ] -thiazole, naphtho [1, 2-d ] thiazole, etc.), thiazoline rings (e.g., thiazoline, 4-methylthiazoline, etc.), oxazole rings (e.g., oxazole, 4-nitrooxazole, etc.), benzoxazoles (e.g., benzoxazole, 4-chlorobenzoxazole, etc.), naphthooxazoles (e.g., naphtho [2, 1-d ] oxazole, naphtho [1, 2-d ] oxazole, etc.), selenazole rings (e.g., selenazole, 4-phenylselenazole, etc.), benzoselenazole rings (e.g., benzoselenazole, 4-chlorobenzselenazole), naphthoselenazole rings (e.g., naphtho [2, 1-d ] selenazole, naphtho [1, 2-d ] selenazole, etc.), (e.g., with a certain ratio of these compounds, 3, 3-dialkylindolenine ring (e.g., 3-dinitroindolenine, 3-diethylindolenine, 3-dimethyl-5-nitroindolenine, etc.), imidazole ring (e.g., 1-alkylimidazole, 1-alkyl-4-phenylimidazole, etc.), pyridine ring (e.g., 2-pyridine, 5-methyl-2-pyridine, etc.), quinoline ring (e.g., 2-quinoline, 3-methyl-2-quinoline, etc.), imidazo [4, 5-b ] quinoxaline ring (e.g., 1, 3-diethylimidazo [4, 5-b ] quinoxaline, etc.), etc. Preferred examples of the 5-to 10-membered heterocyclic ring formed by Y include a 3, 3-dialkylindolenine ring.

M represents a hydrogen atom, a metal, a quaternary ammonium salt or other pharmaceutically acceptable salt. The "pharmaceutically acceptable salt" may be any non-toxic salt formed by using the compound represented by the general formula [ I ]. Examples thereof include, for example, alkali metal salts such as sodium salt, potassium salt and the like; alkaline earth metal salts such as magnesium salt, calcium salt, etc.; organic ammonium salts such as ammonium salts, triethylammonium salts, tributylammonium salts, pyridinium salts, and the like; salts of amino acids such as lysine salts, arginine salts, and the like. Particularly preferred is a sodium salt having lower toxicity to living bodies.

Depending on the type of substituent, the compounds of the present invention may have 1 or more asymmetric carbon atoms. The sulfur atom may serve as an asymmetric center. Optical isomers in optically pure form based on one or more asymmetric carbon atoms, any mixture of the above optical isomers, racemates, diastereomers based on two or more asymmetric carbon atoms, any mixture of the above diastereomers, and the like, are all within the scope of the present invention.

Specific examples of the compounds of the present invention are shown below. However, the scope of the present invention is not limited to the following compounds.

Compound 1

Compound 2

Compound 3

Compound 4

Compound 5

Compound 6

Compound 7

Compound 8

Compound 9

Compound 10

Compound 11

Compound 12

Compound 13

Compound 14

Compound 15

Compound 16

Compound 17

Compound 18

Compound 19

Compound 23

Compound 24

Compound 25

Compound 26

Compound 27

Compound 28

Compound 29

Compound 30

Compound 31

Compound 32

Compound 33

Compound 34

Compound 35

The cyanine dyes represented by the formula [ I ] or [ II ] can be synthesized according to known methods for preparing cyanine dye compounds, such as the methods disclosed in the following documents: the Cyanines and related compounds, F.M. Hamer, John Wiley and Sons, New York, 1964, Cytometry, 11, 416-. Alternatively, they can be semisynthetic by known methods from commercially available cyanine dye compounds. More specifically, they can be synthesized by reacting diaryl compounds with heterocyclic quaternary salts.

The method for preparing the cyanine dye compounds represented by the formula [ I ] or [ II ] is not particularly limited, and these compounds may be synthesized by various synthetic routes. Specific methods for preparing representative compounds of the invention are disclosed in the examples of this specification. Accordingly, cyanine dye compounds within the above general formula can be produced by those skilled in the art by appropriately selecting the starting materials and reagents by the methods described in the examples, and adding suitable changes or modifications to these methods as necessary. In the production, various reactions selected from condensation, addition, oxidation, reduction and the like may be used alone or in combination. These reactions are explained in detail in the literature. For example, various methods and starting compounds described as unit synthesis operations in "Jikken Kagaku Kouza" (published by Maruzen, ltd., including each individual roll available in the first to fourth versions) may be suitably used. In addition, the synthesis of the compounds of the invention is described in detail in the specification of PCT/JP01/06689, the disclosure of which is incorporated herein by reference.

For example, if the above-defined functional groups can be changed in the reaction step or they are not suitable for the reaction step in the preparation, the desired step, for example, a method for protecting or deprotecting the functional group or various treatments such as oxidation, reduction, hydrolysis and the like can be sometimes efficiently carried out by using various methods conventionally used in the field of organic synthetic chemistry. The synthetic intermediate compounds and the target compounds in the above steps can be isolated and purified by conventional purification methods used in organic synthetic chemistry, such as filtration, extraction, washing, drying, concentration, recrystallization, and various chromatographies. The intermediate products of the synthesis can be used in the next step without isolation.

As the active ingredient of the near-infrared fluorescent contrast agent of the present invention, the compounds represented by the general formula [ I ] or [ II ] or salts thereof may be used alone or in combination. More specifically, the active ingredient may be contained in the contrast agent in the form of a suspension or a solution in a solvent such as distilled water for injection, physiological saline, Ringer's solution, or the like. Additives such as pharmaceutically acceptable carriers, excipients, and the like may also be formulated, if desired. Examples of such additives include substances such as pharmaceutically acceptable electrolyte solutions, buffers, detergents, and substances for regulating osmotic pressure, substances for improving stability or solubility such as cyclodextrins, liposomes, and the like. Any additive commonly available in the art may be used. In the case of use as a medicament for clinical use, the near-infrared fluorescent contrast agent of the present invention is preferably synthesized by a sterile process.

The contrast agent of the present invention can be administered by injection, spray, or topical administration such as intravascular administration (intravenous, arterial), oral administration, intraperitoneal administration, transdermal administration, subcutaneous administration, intracapsular administration, or intrabronchial administration. Preferably, the contrast agent is administered into the blood vessel in the form of an aqueous solution, emulsion or suspension.

The dose of the near-infrared fluorescent contrast agent of the present invention is not particularly limited as long as the dose enables the site to be diagnosed to be detected. The dose may be increased or decreased depending on the type of near infrared fluorescence-emitting compound to be used, the age, weight, target organ and the like of the patient to be administered, etc. In general, the dosage expressed in terms of the weight of the compound may be from 0.1 to 100mg/kg body weight, preferably from 0.5 to 20mg/kg body weight.

The contrast agent of the present invention can be suitably used for various animals other than humans. The administration form, administration route, dose and the like can be appropriately selected depending on the body weight and conditions of the target animal.

The compounds of the present invention represented by the above formulas [ I ] and [ II ] have a property of being highly accumulated in tumor tissues. By virtue of this property, the present invention also provides a fluorescent contrast agent capable of specifically imaging tumor tissue. In addition, the compounds of the present invention of this class can be retained in blood vessels for a long period of time, and the fluorescent contrast agents of the present invention can therefore also be used for angiography.

The method for fluorescence imaging of the present invention is characterized by using the near-infrared fluorescent contrast agent of the present invention. The imaging method can be carried out by those skilled in the art according to known methods, and various parameters such as the excitation wavelength and the fluorescence wavelength to be detected can be appropriately determined depending on the kind of the near-infrared fluorescent contrast agent to be administered and the patient to be administered, for optimal imaging and evaluation. The time interval from the administration of the near-infrared fluorescent contrast agent of the present invention to the start of fluorescence imaging according to the present invention may vary depending on the kind of the near-infrared fluorescent contrast agent to be used and the patient to be administered. For example, when a contrast agent comprising a compound of formula [ I ] or formula [ II ] is administered for tumor imaging, the time interval may be from about 10 minutes to 24 hours after administration. If the time interval is too short, the fluorescence emitted in each site is still too strong and the target site cannot be distinguished from other sites. If the time interval is too long, the contrast agent may be excreted from the body. When it is desired to image blood vessels, the compound of formula [ I ] or formula [ II ] can be detected immediately after administration or within about 30 minutes after administration.

For example, fluorescence imaging can be performed as follows. The near-infrared fluorescent contrast agent of the present invention is administered to a patient to be diagnosed, and the patient is exposed to excitation light using a device that generates the excitation light. Then, fluorescence emitted by the near-infrared fluorescent contrast agent and generated by the excitation light is detected using a fluorescence detector. The excitation wavelength may vary depending on the type of near-infrared fluorescent contrast agent used, and is not limited as long as the compound efficiently emits fluorescence in the near-infrared region. Near infrared light having excellent bio-permeability is preferably used. The wavelength of the near infrared fluorescence to be detected also varies depending on the contrast agent used. In general, excitation light having a wavelength of 600-1000nm, preferably 700-850nm may be used, and near-infrared fluorescence having a wavelength of 700-1000nm, preferably 750-900nm may be detected. As the means for generating the excitation light, a conventional excitation light source such as various lasers (e.g., ion laser, dye laser, and semiconductor laser), halogen light source, xenon light source, and the like can be used. Various filters may be used to obtain the optimum excitation wavelength, if desired. In detecting fluorescence, various filters can be used for selecting the fluorescence generated by the near-infrared fluorescent contrast agent.

The detected fluorescence is subjected to data processing as fluorescence information to construct a fluorescence image to be recorded. Examples of the method of forming a fluorescent image include, for example, a method including the steps of: irradiating a target tissue in a wide range, detecting fluorescence by using a CCD camera, and then performing image processing on obtained fluorescence information; a method of using an optical CT apparatus; a method of using an endoscope; or a method using a fundus camera, and the like.

According to the fluorescence imaging method of the present invention, a systemic disease, a tumor, a blood vessel, or the like can be observed without destroying a living body.

Examples

The present invention will be described in more detail below with reference to synthetic examples and test examples. However, the scope of the present invention is by no means limited to the following examples. In these examples, the serial numbers of the compounds correspond to the numbers of the compounds listed in the above chemical structures.

Example 1: synthesis of Compound 1, Compound 2 and Compound 3

The synthetic route of compound 1 is shown below.

Synthesis of intermediate 1

Starting material 1(20.9g, 0.1mol), 2-bromopropionic acid (23.0g, 0.15mol), and o-dichlorobenzene (20ml) were heated and stirred at 140 ℃ for 2 hours. After completion of the reaction, acetone (200ml) was added to the reaction mixture and cooled to room temperature, and then the resulting crystals were filtered to obtain intermediate 1(20.3g, yield: 56%).

Synthesis of intermediate 2

Intermediate 1(10.0g, 28mmol) and 1, 7-diaza-1, 7-diphenyl-1, 3, 5-heptatriene hydrochloride (3.9g, 14mmol) obtained above were dissolved in acetonitrile (70ml) and water (11ml), and triethylamine (8.4g, 91mmol) and acetic anhydride (8.5g, 91mmol) were added to the resulting solution, and the mixture was stirred at room temperature overnight. The reaction mixture was added dropwise to 0.1N hydrochloric acid (900ml), and the precipitated crystals were filtered. The crystals were purified by column chromatography (eluent: dichloromethane: methanol 95: 5-90: 10) to give intermediate 2(2.1g, yield: 12%).

Synthesis of intermediate 3

Intermediate 2(21.0g, 1.5mmol) obtained above, L-aspartic acid-di-tert-butyl ester monohydrate (1.3g, 4.5mmol), 4-dimethylaminopyridine (40mg, 0.3mmol) were dissolved in dichloromethane (50ml), and the solution was cooled on ice. To the resulting solution were added 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) (700mg, 4mmol) and triethylamine (340mg, 3mmol), and the mixture was stirred at 4 ℃ overnight. Methylene chloride (200ml) and 1N hydrochloric acid (200ml) were added to the reaction mixture, and the methylene chloride layer was extracted and washed with a saturated sodium chloride solution (200ml), followed by drying over sodium sulfate. The solvent was evaporated under reduced pressure and purified by column chromatography (eluent: ethyl acetate: methanol 95: 5-80: 20) to give intermediate 3(1.1g, yield: 64%).

Synthesis of Compound 1, Compound 2 and Compound 3

Intermediate 3(500mg, 0.5mmol) was dissolved in trifluoroacetic acid (5ml), reacted at 4 ℃ overnight, and then the trifluoroacetic acid was evaporated under reduced pressure. The crystals were collected by filtration, and then washed with water and ethyl acetate to give compound 1(390mg, yield: 90%).

Compound 1 was purified by column chromatography (eluent: methanol) using Sephadex (LH-20, Pharmacia) to give compound 2.

Compound 1 was placed on ion exchange resin column CR 11(Mitsubishi Chemical, co., Ltd.) to give compound 3.

Compound 1

¹H-NMR(CD₃OD)δ1.98(s，12H)，2.70(d，J＝7.2Hz，4H)，2.80(t，J＝7.2Hz，4H)，3.30(MeOH)，4.50(t，J＝7.2Hz，4H)，4.60(t，J＝7.2Hz，2H)，4.80(H₂O)，6.40(d，J＝13.2Hz，2H)，6.63(dd，J＝13.2，13.2Hz，2H)，7.40-7.50(m，2H)，7.58-7.66(m，5H)，7.95-8.07(m，6H)，8.20(d，J＝7.2Hz，2H)

Compound 2

¹H-NMR(CD₃OD)δ1.99(s，12H)，2.72(d，J＝7.2Hz，4H)，2.80(t，J＝7.2Hz，4H)，3.30(MeOH)，4.50(t，J＝7.2Hz，4H)，4.60(t，J＝7.2Hz，2H)，4.80(H₂O)，6.38(d，J＝13.2Hz，2H)，6.61(dd，J＝13.2，13.2Hz，2H)，7.40-7.50(m，2H)，7.58-7.67(m，5H)，7.96-8.07(m，6H)，8.21(d，J＝7.2Hz，2H)

Compound 3

¹H-NMR(CD₃OD)δ1.98(s，12H)，2.56-2.65(m，4H)，2.75-2.85(m，4H)，3.30(MeOH)，4.45-4.50(m，4H)，4.80(H₂O)，6.20(d，J＝13.2Hz，2H)，6.65(dd，J＝13.2，13.2Hz，2H)，7.40-7.50(m，2H)，7.58-7.70(m，5H)，7.95-8.07(m，6H)，8.20(d，J＝7.2Hz，2H)

Example 2: synthesis of Compound 5

Compound 5 was synthesized from intermediate 1 and 1, 7-diaza-5-methyl-1, 7-diphenyl-1, 3, 5-heptatriene monohydrate according to a method analogous to that of Compound 1.

¹H-NMR(CD₃OD)δ2.00(s，12H)，2.44(s，3H)，2.73(d，J＝7.2Hz，4H)，2.82(t，J＝7.2Hz，4H)，3.31(MeOH)，4.50(t，J＝7.2Hz，4H)，4.69(t，J＝7.2Hz，2H)，4.88(H₂O)，6.41(d，J＝13.2Hz，2H)，6.65(d，J＝13.2Hz，2H)，7.43-7.50(m，2H)，7.58-7.67(m，4H)，7.95-8.05(m，4H)，8.10-8.27(m，4H)

Example 3: synthesis of Compound 6

Compound 6 was synthesized from intermediate 1 and 1, 7-diaza-5-methyl-1, 7-diphenyl-1, 3, 5-heptatriene monohydrate following a procedure similar to compound 1, except that: instead of L-aspartic acid-di-tert-butyl ester monohydrate, L-glutamic acid-di-tert-butyl ester monohydrate was used.

¹H-NMR(CD₃OD)δ1.80-2.15(m，4H)，2.01(s，12H)，2.28(t，J＝7.2Hz，4H)，2.44(s，3H)，2.82(t，J＝7.2Hz，4H)，3.31(MeOH)，4.40-4.50(m，2H)，4.51(t，J＝7.2Hz，4H)，4.88(H₂O)，6.42(d，J＝13.2Hz，2H)，6.65(d，J＝13.2Hz，2H)，7.42-7.50(m，2H)，7.57-7.67(m，4H)，7.95-8.05(m，4H)，8.10-8.27(m，4H)

Example 4: synthesis of Compound 7

Compound 7 was synthesized from 2, 3, 3-trimethylindolenine in analogy to compound 1.

¹H-NMR(CD₃OD)δ1.70(s，12H)，2.05-2.13(m，4H)，2.55(t，J＝7.2Hz，4H)，2.78-2.92(m，4H)，3.30(MeOH)，4.10(t，J＝7.2Hz，4H)，4.89(H₂O)，6.45(d，J＝13.2Hz，2H)，6.50(J＝13.2Hz，2H)，7.29-7.50(m，8H)，7.92(dd，J＝13.2，13.2Hz，2H)

Example 5: synthesis of Compound 8

Compound 8 was synthesized from 2, 3, 3-trimethylindolenine in analogy to compound 1, except that: 1, 7-diaza-5-methyl-1, 7-diphenyl-1, 3, 5-heptatriene monohydrochloride was used instead of 1, 7-diaza-1, 7-diphenyl-1, 3, 5-heptatriene monohydrate.

¹H-NMR(CD₃OD)δ1.70(s，12H)，1.72-1.90(m，8H)，2.35-2.39(m，7H)，2.73-2.84(m，4H)，3.30(MeOH)，4.08(t，J＝7.2Hz，4H)，4.66(t，J＝7.2Hz，2H)，4.89(H₂O)，6.33(d，J＝13.2Hz，2H)，6.63(d，J＝13.2Hz，2H)，7.18-7.50(m，8H)，8.05(dd，J＝13.2，13.2Hz，2H)

Example 6: synthesis of Compound 9

Compound 9 was synthesized from 6-phenyl-2, 3, 3-trimethylindolenine (synthesized according to the procedure described in U.S. Pat. No. 6,004,536) in analogy to Compound 1.

¹H-NMR(CD₃OD)δ1.75(s，12H)，2.05-2.15(m，4H)，2.45-2.55(m，4H)，2.75-2.84(m，4H)，3.30(MeOH)，4.20(t，J＝7.2Hz，4H)，4.80(H₂O)，6.38(J＝13.2Hz，2H)，6.62(J＝13.2Hz，2H)，7.43-7.70(m，17H)，7.95(dd，J＝13.2，13.2Hz，2H)

Example 7: synthesis of Compound 10

Compound 10 was synthesized from 6-bromo-2, 3, 3-trimethyl-indolenine in analogy to compound 1.

¹H-NMR(CD₃OD)δ1.68(s，12H)，2.00-2.15(m，4H)，2.40-2.55(m，4H)，2.77-2.92(m，4H)，3.30(MeOH)，4.08(t，J＝7.2Hz，4H)，4.82(m，2H)，6.38(J＝13.2Hz，2H)，6.65(J＝13.2Hz，2H)，7.30-7.40(m，4H)，7.50-7.72(m，3H)，7.90-8.02(m，2H)

Example 8: synthesis of Compound 11

Compound 11 was synthesized from 5-phenyl-2, 3, 3-trimethyl-indolenine in analogy to compound 1.

¹H-NMR(CD₃OD)δ1.78(s，12H)，2.39(s，3H)，2.70-2.84(m，8H)，3.30(MeOH)，4.30-4.46(m，4H)，4.60-4.68(m，2H)，6.39(J＝13.2Hz，2H)，6.66(J＝13.2Hz，2H)，7.30-7.48(m，9H)，7.56-7.72(m，3H)，8.05(J＝13.2Hz，13.2Hz)

Example 9: synthesis of Compound 13 and Compound 14

The synthetic routes for compound 13 and compound 14 are shown below.

An intermediate compound (375mg) prepared by reacting 5-sulfo-2, 3, 3-trimethylindolenine (prepared according to the method described in Japanese unexamined patent application publication (Hei) No. 2-233658) and 1, 7-diaza-1, 7-diphenyl-1, 3, 5-heptatriene monohydrochloride in methanol in the presence of triethylamine and acetic anhydride was dissolved in 5ml of methanol and then applied to a column (Organo, eluent: methanol) packed with an ion exchange resin IRC-50. The solvent is evaporated to give the carboxylic acid in protic form. The resulting product was dissolved in 3ml of dimethylformamide, and 338mg (1.2mmol) of dibutyl aspartate hydrochloride, 24mg (0.2mmol) of dimethylaminopyridine and 121mg (1.2mmol) of triethylamine were added to the solution, and then the mixture was cooled on an ice bath. To the mixture was added 230mg (2mmol) of Hydroxysuccinimide (HOSI) and 288mg (1.4mmol) of N, N-dicyclohexyl-carbodiimide (DCC), and the resulting mixture was stirred overnight. To the reaction mixture was added 200ml of a mixed solvent of ethyl acetate/hexane (1: 1), and crystals precipitated were collected by filtration. The crystals were purified by column chromatography (eluent: dichloromethane: methanol-10: 1-2: 1) to give a diamide compound (135mg) and a monoamide compound (94 mg).

The resulting diamide compound (120mg) and monoamide compound (60mg) were dissolved in 2ml of trifluoroacetic acid, respectively, and the mixture was stirred at room temperature for 1 hour. The reaction mixture was dissolved in water/methanol (1/1(v/v)), and then purified by column chromatography using Sephadex (LH-20, Pharmacia, eluent: methanol). The resulting crystals were dissolved in a small amount of methanol, and a saturated solution of potassium acetate in methanol was added to the solution. The precipitated crystals were collected by filtration to give compound 13(35mg, yield 7%) and compound 14(15mg, yield 5%).

Compound 13

¹H-NMR(D₂O)δ1.73(s，12H)，2.50-2.65(m，4H)，2.68-2.73(m，4H)，4.28-4.38(m，4H)，4.39-4.50(m，2H)，4.90(D₂O)，6.47(d，J＝13.2Hz，2H)，6.74(t，J＝13.2Hz，2H)，7.40-7.50(m，2H)，7.60(t，J＝13.2Hz，1H)，7.80-8.05(m，6H)

Compound 14

¹H-NMR(D₂O)δ1.65(s，6H)，1.70(s，6H)，2.40(d，J＝7.2Hz，2H)，2，58(t，J＝7.2Hz，2H)，2.70(t，J＝7.2Hz，2H)，4.18-4.30(m，4H)，4.90(D₂O)，6.18(d，J＝13.2Hz，1H)，6.34(d，J＝13.2Hz，1H)，6.48-6.62(m，2H)，7.20(d，J＝7.2Hz，1H)，7.30(d，J＝7.2Hz，1H)，7.48(t，J＝13.2Hz，1H)，7.68-7.95(m，6H)

Example 10: synthesis of Compound 15

The synthetic route for compound 15 is shown below.

The starting material (41.8g, 0.2mol) was dissolved in concentrated sulfuric acid (156ml, 2.9mol) and reacted at 140 ℃ for 1 hour, and then the mixture was cooled to 80 ℃. After the resulting solution was added to ice water (300ml), a solution of sodium hydroxide (96.6g, 2.4mol) in water (100ml) was carefully added to the above mixture. The precipitated crystals were collected by filtration and washed with water (120 ml). To the resulting crude crystals were added water (300ml) and methanol (100ml), and the mixture was refluxed for 30 minutes with stirring, and then cooled to room temperature. The resulting crystals were collected by filtration and washed with water (100ml) and methanol (120ml) to give intermediate 5(37.9g, yield: 66%).

Compound 15 was prepared from intermediate 5 following a procedure analogous to compound 13.

¹H-NMR(CD₃OD)δ2.00(s，12H)，2.72(d，J＝7.2Hz，4H)，2.82(t，J＝7.2Hz，4H)，3.30(MeOH)，4.58(t，J＝7.2Hz，4H)，4.70(t，J＝7.2Hz，4H)，4.86(H₂O)，6.42(d，J＝13.2Hz，2H)，6.62(dd，J＝13.2，13.2Hz，2H)，7.62-7.70(m，3H)，7.95-8.12(m，6H)，8.28(d，J＝7.2Hz，2H)，8.42(s，2H)

Example 11: synthesis of Compound 23

The synthetic route for compound 23 is shown below.

Synthesis of intermediate 6

5-sulfo-2, 3, 3-trimethylindolenine (prepared according to the method described in Japanese unexamined patent application publication (Hei) No. 2-233658) (24.0g, 0.1mol), 2-bromopropionic acid (23.0g, 0.15mol) and triethylamine (10.1g, 0.1mol) were heated and stirred at 160 ℃ for 6 hours. After completion of the reaction, methanol (200ml) was added to the reaction mixture, cooled to room temperature, and then the resulting crystals were collected by filtration to give intermediate 6(6.0g, yield: 19.3%).

Synthesis of Compound 23

The intermediate 1(3.1g, 10mmol) obtained above and 1, 7-diaza-1, 7-diphenyl-4-methyl-1, 3, 5-heptatriene monohydrochloride (Japanese unexamined patent application publication (Hei) No. 8-295658) (1.5g, 5mmol) were dissolved in methanol (20ml), and triethylamine (2.5g, 25mmol) and acetic anhydride (4.6g, 45mmol) were added to the resulting solution, and the mixture was stirred at room temperature for 3 hours. To the reaction mixture was added sodium acetate (3.3g, 33mmol), followed by stirring at room temperature for 30 minutes. The resulting crystals were collected by filtration and washed with methanol (20ml) to give compound 23(2.0g, yield: 50.0%).

¹H-NMR(CD₃OD)δ(ppm)1.60(s，12H)，2.30(s，3H)，2.60(t，4H，J＝7.2Hz)，4.20(t，4H，J＝7.2Hz)，6.25(d，2H，J＝14.5Hz)，6.55(dd，2H，14.5，14.5Hz)，7.25(d，2H，J＝7.0Hz)，7.70-7.80(m，4H)，8.00(dd，2H，J＝14.5，14.5Hz)

Example 12: synthesis of Compound 25 and Compound 26

The synthetic routes for compound 25 and compound 26 are shown below.

Synthesis of intermediate 7

Intermediate 7(16.6g) was synthesized from 5-sulfo-2, 3, 3-trimethylindolenine and bromoacetic acid in analogy to intermediate 6.

Synthesis of Compound 25

Compound 25(15.0g) was synthesized from intermediate 7 and intermediate 8 (prepared according to the method described in zh.org.khim., 13, pp.1189-1192, 1977) in analogy to compound 23.

MS (FAB-, glycerol) m/z 844

Synthesis of Compound 26

Compound 25(4.2g, 5mmol) and triethylamine (1.0g) were added to water (20ml), and o-mercaptobenzoic acid (0.93g, 6mmol) was added to the resulting solution, followed by stirring at room temperature for 1 hour. To the resulting mixture was added potassium acetate, followed by ethanol (20ml), and the resulting crystals were filtered to give compound 26(1.3g, yield: 27%).

MS (FAB-, glycerol) m/z 962

Example 13: synthesis of Compound 32

The synthetic route for compound 32 is shown below.

Synthesis of intermediate 9

4-bromophenylhydrazine monohydrochloride (73.8g, 0.33mmol) and 3-methyl-2-butanone (33.2g, 0.40mmol) were dissolved in ethanol (450ml), and concentrated sulfuric acid (7.5ml) was added to the resulting solution, followed by reflux with stirring for 8 hours. After the mixture was cooled to room temperature, the solution was concentrated to 100ml under reduced pressure. To the residue were added water (400ml) and ethyl acetate (400ml), and then the pH of the aqueous layer was adjusted to 7-8 with sodium hydroxide solution. The resulting solution was extracted with ethyl acetate, washed with a saturated sodium chloride solution, and then dried over anhydrous sodium sulfate. The resulting residue was purified by silica gel column chromatography (eluent: hexane: ethyl acetate 5: 1-1: 1) to give intermediate 9(58.6g, 76% yield) as a brown liquid.

Synthesis of intermediate 10

Intermediate 9(4.76g, 20mmol) and thiopheneboronic acid (3.84g, 30mmol) were added to dimethylamide (50ml), and to the resulting solution were added palladium tetraphenylphosphine (1.16g, 9mmol) and cesium chloride (13.3g, 40mmol), followed by heating under nitrogen and stirring at 100 ℃ for 4 hours. After water (200ml) was added, the mixture was extracted with ethyl acetate (200ml), which was washed with saturated sodium chloride solution, and the organic layer was dried over anhydrous sodium sulfate, followed by evaporation under reduced pressure. The residue was purified by silica gel column chromatography (eluent: hexane: ethyl acetate 2: 1-1: 1) to give intermediate 10(2.8g, yield: 58%) as a brown solid.

Synthesis of intermediate 11

Intermediate 10(1.40g, 6mmol) and triethylamine (0.59g, 6mmol) were added to dimethylformamide (3ml), and 2-chloroethanesulfinylchloride (1.42g, 9mmol) was added dropwise to the mixture under ice-cooling. After stirring at room temperature was continued for 30 minutes, a solution of sodium hydroxide (0.23g, 6mmol) in water (2ml) was added to the solution, followed by further stirring at room temperature for 1 hour. To the mixture was added ethyl acetate, and the upper layer was decanted. The residue was dried to give intermediate 11. This intermediate 11 was used in the next reaction without purification.

Synthesis of Compound 32

The intermediate 11 and 1, 7-diaza-1, 7-diphenyl-1, 3, 5-heptatriene monohydrochloride obtained as above were dissolved in methanol (5ml), triethylamine (160mg, 2mmol) and anhydrous acetic acid (230mg, 2mmol) were added to the resulting solution, and the mixture was stirred at room temperature for 7 hours. To the mixture was added ethyl acetate (20ml), and the precipitated crystals were collected by filtration and washed with ethyl acetate (10 ml). The crystals were dissolved in methanol (10ml), and a saturated solution of saturated potassium acetate in methanol (10ml) was added to the solution. The precipitated crystals were collected by filtration and washed with methanol (5 ml). The crystals were purified by Sephadex LH-20 (eluent: methanol) to give compound 32(15mg, yield: 2%, calculated from intermediate 2).

¹H-NMR(CD₃OD)δ(ppm)1.75(s，12H)，3.25(t，4H，J＝7.2Hz)，4.50(t，4H，J＝7.2Hz)，6.40(d，2H，J＝14.5Hz)，6.63(dd，2H，14.5，14.5Hz)，7.07-7.12(m，2H)，7.33-7.45(m，6H)，7.53-7.75(m，5H)，7.96(dd，2H，J＝14.5，14.5Hz)

MS (FAB-, Glycerol) m/z 760

Example 14: synthesis of Compound 33

The synthetic route for compound 33 is shown below.

Synthesis of intermediate 12

Intermediate 12(3.6g, yield: 77%) was synthesized from intermediate 9 and dihydroxyphenylborane according to a method similar to intermediate 10.

Synthesis of intermediate 13

Intermediate 12(1.40g, 6mmol) and 1, 4-butane sultone (1.22g, 9mmol) were dissolved in dimethylacetamide (2ml) and the solution was stirred at 135 ℃ for 5 hours. To the solution was added ethyl acetate (20ml), followed by cooling to room temperature. The precipitated crystals were collected by filtration and then washed with ethyl acetate to give intermediate 13(10ml) (1.84g, yield: 84%).

Synthesis of Compound 33

Intermediate 13(1110mg, 3mmol) and 1, 7-diaza-1, 7-diphenyl-1, 3, 5 heptatriene monohydrochloride (285mg, 1mmol) were dissolved in methanol (5ml), and triethylamine (480mg, 5mmol) and anhydrous acetic acid (670mg, 7mmol) were added to the resulting solution, followed by stirring at room temperature for 7 hours. Ethyl acetate (10ml) was added to the reaction mixture, and the precipitated crystals were collected by filtration and then washed with ethyl acetate (10 ml). The crystals were dissolved in methanol (5ml) and a saturated solution of potassium acetate in methanol (10ml) was added, and the precipitated crystals were filtered and washed with 5 ml. The crystal was purified with Sephadex LH-20 (diluent: methanol) to give compound 33(250mg, yield: 30%).

¹H-NMR(CD₃OD)δ(ppm)1.80(s，12H)，1.95-2.05(m，8H)，2.90(t，4H，J＝7.2Hz)，4.20(t，4H，J＝7.2Hz)，6.38(d，2H，J＝14.5Hz)，6.62(dd，2H，14.5，14.5Hz)，7.30-7.48(m，8H)，7.60-7.74(m，9H)，7.93(dd，2H，J＝14.5，14.5Hz)

MS (FAB-, nitrobenzyl alcohol) m/z 803

Example 15: synthesis of Compound 34

Compound 34(15mg) was synthesized from intermediate 9 and 4-methylmercaptophenylboronic acid in analogy to compound 33.

¹H-NMR(CD₃OD)δ(ppm)1.68(s，12H)，1.95-2.10(m，8H)，2.50(s，6H)，3.00(t，4H，J＝7.2Hz)，4.10(t，4H，J＝7.2Hz)，6.30(d，2H，J＝14.5Hz)，6.62(dd，2H，14.5，14.5Hz)，7.20-7.70(m，19H)

Example 16: synthesis of Compound 35

The synthetic route for compound 35 is shown below.

Synthesis of intermediate 14

25.0g of 3-aminobiphenyl (0.15mol) were added to 100ml of trifluoroacetic acid, and the mixture was cooled to an internal temperature of 0 ℃. To this mixture was added dropwise a solution of 10.2g of sodium nitrite (0.15mol) in 100ml of water, while the temperature of the reaction mixture was kept below 5 ℃. After completion of the dropwise addition, the mixture was stirred at the same temperature for 15 minutes, and then a solution of 100g of tin chloride (0.54mol) in 50ml of concentrated hydrochloric acid was added to the mixture while the temperature of the reaction mixture was kept below 10 ℃. After completion of the dropwise addition, 250ml of water was added, and the precipitated crystals were collected by filtration and washed with 200ml of dichloromethane. The resulting intermediate 14 was dried and used in the synthesis of intermediate 15 without purification.

Synthesis of intermediate 15

The intermediate 14 (total amount) obtained as above and 12.9g of 3-methyl-2-butanone (0.15mol) were added to 140ml of acetic acid, and the mixture was heated with stirring for 2.5 hours. After cooling the mixture to room temperature, the precipitated crystals were removed by filtration and the filtrate was concentrated under reduced pressure to 1/4 volumes. To the residue were added 300ml of water and 300ml of ethyl acetate, and insoluble precipitates were removed by filtration through celite. The filtrate was extracted with ethyl acetate (300ml, 200 ml. times.2), and the extract was washed with a saturated sodium hydrogencarbonate solution and saturated brine, then dried over sodium sulfate, and the solvent was removed by evaporation under reduced pressure. The residue is purified by chromatography on silica gel (eluent: hexane: ethyl acetate 3: 1-2: 1). The obtained crystals were recrystallized from 50ml of hexane to obtain intermediate 15(1.3g, yield: 4%).

Synthesis of Compound 35

Compound 35(65mg) was synthesized from intermediate 15 in a similar manner to intermediate 13 and compound 33.

MS (FAB-, glycerol) m/z 842.804

¹H-NMR(D₂O)δ(ppm)1.70(s，12H)，1.90-2.00(m，8H)，2.90(t，4H，J＝7.2Hz)，4.10(t，4H，J＝7.2Hz)，6.22(d，2H，J＝14.5Hz)，6.55(dd，2H，14.5，14.5Hz)，7.30-7.60(m，17H)，7.77(dd，2H，J＝14.5，14.5Hz)

Test example 1: fluorescence imaging test

Tumor tissue sections of mouse colon cancer (colon 26 carcinoma) were subcutaneously transplanted into the left breast of BALB/c hairless mice (5 weeks old, clean Japan, Inc.). After 10 days, when the tumor grew to about 8mm, the mice were used in the experiment. As a fluorescence excitation light source, a titanium sapphire laser was used. A ring type light guide (Sumita Optical Glass Co.) was used, in which the dispersion of irradiation was within 10%, thereby uniformly exposing the test mice to the light of the laser. The irradiation power output was adjusted so that the power was about 40. mu.W/cm near the skin surface of the mouse². Fluorescence was excited at the maximum excitation wavelength of each compound, and then the fluorescence emitted in the mice was detected and photographed by short-wave cut filters (IR84, IR86, IR88, Fuji Photo Film co., LTD.) with a CCD camera (C4880, Hamamatsu Photonics K.K.). The choice of the cut-off filter should be adapted to the excitation wavelength of the compound. The exposure time was adjusted depending on the fluorescence intensity of each compound. Compound 2(0.5mg/ml) as a test compound was dissolved in physiological saline or phosphate buffer (pH7.4) and then administered to mice at a dose of 5.0mg/Kg through the tail vein. At a given time after administration of the test compound, mice were anesthetized with ether and fluorescence plots of the entire body of the mice were performedThe image is taken. For comparison, ICG (5mg/kg, i.v.) and the following compound (Compound A) were administered separately, followed by imaging in the same manner as above. The results are shown in FIGS. 1-3.

Compound 2 gave clear images of the tumor in a shorter time after administration compared to the reference compound. Within 1 hour of administration of the reference compound, the location of the tumor was unclear. Compound 2 successfully gave clear images of tumors 10-30 minutes after dosing and showed high efficacy as a fluorescent contrast agent (figure 1).

Test example 2: fluorescence imaging test

Tumor-bearing mice were prepared in the same manner as in test example 1, and the irradiation conditions were the same as described in test example 1.

Compound 5, compound 7, and compound 10 were used as test compounds. Each test compound (0.5mg/ml) was dissolved in physiological saline or phosphate buffer (pH7.4), and then administered to mice at a dose of 5.0mg/Kg through the tail vein. For comparison, the following compound (compound B, 5mg/kg, i.v.) was administered to mice.

Light is generated using a tunable, pulsed solid-state laser system consisting of an Optical Parametric Oscillator (OPO) driven by the third harmonic of a Nd: Yag laser (Coherent Inc.). Excitation wavelengths λ ex ═ 740nm were selected and directed to tumor-bearing hairless mice with optical fibers. Dye-specific fluorescence emission was imaged at different times after dye administration using a filter combination (Corion) and an enhanced CCD camera (RoperScientific) (fig. 4). Fluorescence imaging was performed prior to dosing and 1 minute, 10 minutes, 30 minutes, 60 minutes, 2 hours, 4 hours, 24 hours after intravenous dosing of the dye through the lateral tail vein at a standard dose of 5 mg/kg. The body temperature of the animal was maintained at 38 ℃ with a heating pad for the first 60 minutes. The fluorescence imaging properties of each compound were compared in a hairless mouse tumor model. The results are shown in FIGS. 5-8. Compound 5, compound 7 and compound 10 gave clear images of tumors in a shorter time after administration compared to the reference compound (compound B). Within 1 hour of administration of the reference compound, the location of the tumor was unclear (fig. 8). The compounds of the invention successfully gave clear images of tumors 10-30 minutes after administration (FIGS. 5-7) and showed high efficacy as fluorescent contrast agents.

Industrial applicability of the invention

The near-infrared fluorescent contrast agent of the present invention can emit near-infrared fluorescence by excitation light. The near-infrared fluorescence is excellent in permeability of biological tissues, and the contrast agent is thus capable of detecting lesions in deeper parts of a living body.

Claims (10)

1. A composition for fluorescence imaging, comprising a compound represented by the following formula [ I ] or a pharmaceutically acceptable salt thereof, and pharmaceutically acceptable carriers and excipients:

wherein:

R¹、R²、R⁷and R⁸Independently of one another, represents substituted or unsubstituted C_1-10Alkyl or substituted or unsubstituted arylAnd R is¹And R²And/or R⁷And R⁸Can be connected together to form a ring;

R³、R⁴、R⁵、R⁶、R⁹、R¹⁰、R¹¹and R¹²Independently of one another, represents a hydrogen atom, a substituted or unsubstituted C_1-6Alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, halogen, cyano, carboxyl or sulfo, and R³、R⁴、R⁵、R⁶、R⁹、R¹⁰、R¹¹And R¹²Can be connected together to form a ring;

m¹represents 0 or 1;

m²represents 0 or 1;

m³represents 0 or 1;

L¹、L²、L³、L⁴、L⁵、L⁶and L⁷Independently represents a substituted or unsubstituted methine group, with the proviso that: when two or more methines have a substituent, the substituents may be linked together to form a ring;

m represents a hydrogen atom, a metal or a quaternary ammonium salt;

n represents an integer of 1 to 7 required for neutralizing charge;

X²represents substituted or unsubstituted C_1-15Alkyl or substituted or unsubstituted aryl; and

X¹represents a group represented by the following formula (i):

wherein Y is₁And Y₂Independently represents a substituted or unsubstituted divalent linking group, X¹And X²Having a total of 2-4 carboxyl groups.

2. The composition of claim 1, wherein m is¹、m²And m³Respectively 1.

3. The composition of claim 1, wherein X¹And X²Independently represents a group represented by the following formula (i):

wherein Y is₁And Y₂Independently represent a substituted or unsubstituted divalent group.

4. The composition of claim 1, wherein R³、R⁴、R⁵、R⁶、R⁹、R¹⁰、R¹¹And R¹²At least one of which is a substituted or unsubstituted aryl group or a substituted or unsubstituted heteroaryl group.

5. The composition of claim 1, wherein Y₁Represents- (CH)₂)_pCONH-, wherein p represents an integer of 1 to 4 and Y₂Represents- (CH)₂) -or (CH)₂)₂-。

6. The composition of any one of claims 1 to 5, wherein the fluorescence imaging is for tumor imaging.

7. The composition of any one of claims 1 to 5, wherein the fluorescence imaging is for angiography.

8. Use of a composition according to any one of claims 1 to 5 for the preparation of a medicament for fluorescence imaging of a living body.

9. The use of claim 8, wherein the medicament is for tumor imaging.

10. The use of claim 8, wherein the medicament is for angiography.