JP2003160558A - Near-infrared fluorescent contrast agent and fluorescent imaging - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a fluorescent contrast agent having high water solubility and good transmissibility to biological tissues, which enables the detection of lesions that locate in the deep part of the body. <P>SOLUTION: A sodium salt represented by formula (1) is provided. A near- infrared fluorescent contrast agent containing the sodium salt used for tumor imaging or angiography is also provided. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a near-infrared fluorescence contrast agent and fluorescence imaging using the contrast agent.

[0002]

2. Description of the Related Art In treating a disease, it is extremely important to detect morphological and functional changes caused in the body by the disease at an early stage of the disease.
The location and size of the tumor are important determinants of effective treatment planning, especially when treating cancer. Methods already known for this purpose include biopsy by puncture and the like, and imaging diagnostics such as X-ray imaging, MRI, ultrasound imaging and the like. Although biopsy is effective for definitive diagnosis, it also puts a heavy burden on the subject and is not suitable for tracking the time course of lesions. X-ray imaging and MRI necessarily expose the subject to radiation and electromagnetic waves. Furthermore, the conventional imaging diagnosis as described above requires complicated operation and long time for measurement and diagnosis. The size of the equipment used for this purpose also makes these methods difficult to use in operation.

One of imaging diagnostics is fluorescence imaging (Lipspn RL et al., J. Natl. Cancer Ins.
t., 26, 1-11 (1961)). In this method, a substance that emits fluorescence upon irradiation with excitation light having a specific wavelength is used as a contrast agent. For example, irradiate the body with excitation light from the outside,
Fluorescence emitted from the fluorescent contrast agent in the body is detected. Such a fluorescent contrast agent is, for example, a porphyrin compound that accumulates in tumors, and is used for photodynamic therapy (PDT) like hematoporphyrin. Other examples include photofrin and benzoporphyrin (Lipspn
RL et al., Supra; Meng TS et al., SPIE, 164
1, 90-98 (1992); see WO84 / 04665, etc.). These compounds were originally used in PDT and have phototoxicity, which is the performance required by PDT. Therefore,
These are not desirable as diagnostic agents.

On the other hand, a retinal circulation microangiography method using known fluorescent dyes such as fluorescein, fluoresamine and riboflavin is known (US Pat. No. 4,945,239).
issue). These fluorescent dyes emit fluorescence in the visible light region of 400-600 nm. In this region, the light transmission through living tissue is so low that it is almost impossible to detect lesions in the deep part of the body.

Further, it has been reported that cyanine compounds including indocyanine green (hereinafter abbreviated as ICG) used for measuring liver function and cardiac output are used as fluorescent contrast agents (Haglund MM et al. ., Neurosurge
ry, 35, 930 (1994), Li, X. et al., SPIE, 2389, 789-
797 (1995)). Cyanine compounds are in the near infrared region (700-13
Absorption at 00 nm).

Near-infrared light is highly transparent through living tissue and can penetrate a skull of about 10 cm. Therefore, it is gaining more and more attention in the field of clinical medicine. For example, an optical CT method using optical transmission of a medium has received attention in the clinical field as a new science and technology. This is because near-infrared light can pass through a living body and can be used to monitor oxygen concentration in the living body and its circulation.

Cyanine compounds emit fluorescence in the near infrared region. Fluorescence in this region can penetrate biological tissues and has potential as a fluorescent contrast agent. Various cyanine compounds have been developed in recent years and are being tried as fluorescent contrast agents (WO96 / 17628, WP97 / 13490, etc.). However, there is no contrast agent having the ability to distinguish normal tissue from diseased tissue (selectivity with respect to the imaging target site), as well as sufficient water solubility and sufficient safety for the living body.

[0008]

DISCLOSURE OF THE INVENTION It is therefore an object of the present invention to provide a fluorescent contrast agent. The contrast agent of the present invention has low toxicity and excellent water solubility. Furthermore, it emits near-infrared fluorescence that can penetrate through biological tissue, allowing specific imaging of tumors and / or blood vessels. Another object of the present invention is to provide a fluorescence imaging method using the near infrared fluorescence contrast agent. The present invention is based on the finding that a fluorescent contrast agent having high water solubility is provided by introducing three or more sulfonic acid groups into a cyanine dye compound. It was also found that a fluorescence imaging method can be established using this contrast agent. .

That is, the present invention provides the following: (1) A compound having 3 or more sulfonic acid groups in the molecule and represented by the formula [I]:

[Chemical 2] {In the formula, R ¹ and R ² are the same or different and each is a substituted or unsubstituted alkyl group; Z ¹ and Z ² are each a substituted or unsubstituted fused benzo ring or fused naphtho ring; Is a necessary non-metal atom; r is 0, 1 or 2; L ¹ -L ⁷ are the same or different and are respectively substituted or unsubstituted methine groups, provided that when r is 2, two L ⁶ and L ^{7 which} are respectively present are the same or different; X and Y are the same or different and each is a group represented by the following formula:

[Chemical 3] (Wherein R ³ and R ⁴ are the same or different and each is a substituted or unsubstituted alkyl group)} or a pharmaceutically acceptable salt thereof. (2) The near-infrared fluorescent contrast agent according to (1) above, which does not contain a carboxylic acid group in the molecule. (3) The above (1) or (2), wherein r is 1 in the formula [I].
Near infrared fluorescent contrast agent. (4) The near-infrared fluorescent contrast agent according to any one of (1) to (3) above, which contains 4 or more sulfonic acid groups in the molecule. (5) The near-infrared fluorescent contrast agent according to any one of (1) to (4) above, which contains 10 or less sulfonic acid groups in the molecule. (6) The near-infrared fluorescent contrast agent according to any one of (1) to (4) above, which contains 8 or less sulfonic acid groups in the molecule. (7) The near-infrared fluorescent contrast agent according to any one of (1) to (6) above, wherein the pharmaceutically acceptable salt is a sodium salt. (8) The near-infrared fluorescent contrast agent according to any one of (1) to (7) above for tumor imaging and / or angiography. (9) Formula [I having three or more sulfonic acid groups in the molecule
Sodium salt of compound I]:

[Chemical 4] (In the formula, R ¹ , R ² , L ¹ -L ⁷ , X and Y are as defined above, and R ⁵ to R ¹⁶ are the same or different, and each is a hydrogen atom, a sulfonic acid group, a carboxyl group, Hydroxyl group, alkyl (sulfoalkyl) amino group, bis (sulfoalkyl)
Amino group, sulfoalkoxy group, (sulfoalkyl) sulfonyl group or (sulfoalkyl) aminosulfonyl group) However, the following compounds are excluded.

[Chemical 5]

[Chemical 6] (10) In formula [II], R ¹ and R ² are each a lower alkyl group having 1 to 5 carbon atoms substituted with a sulfonic acid group, and X and Y are the same or different and Expression of:

[Chemical 7] The sodium salt of the above (9), which is a group represented by the formula: wherein R ¹⁷ and R ¹⁸ are unsubstituted lower alkyl groups having 1 to 5 carbon atoms. (11) The sodium salt of the above (10) represented by the following formula:

[Chemical 8] (12) Sodium salt of the compound of formula [III-1] having 3 or more sulfonic acid groups in the molecule:

[Chemical 9] (In the formula, L ¹ -L ⁷ are as defined above; R ¹⁹ and R ²⁰
Is a lower alkyl group having 1 to 5 carbon atoms, which is substituted with a sulfonic acid group; R ²¹ -R ²⁸ are the same or different and each is a hydrogen atom, a sulfonic acid group, a carboxyl group, a hydroxyl group, an alkyl group. A (sulfoalkyl) amino group, a bis (sulfoalkyl) amino group, a sulfoalkoxy group, a (sulfoalkyl) sulfonyl group or a (sulfoalkyl) aminosulfonyl group; X ′ and Y ′ are the same or different and formula:

[Chemical 10] (In the formula, R ¹⁷ and R ¹⁸ have the same meanings as defined above.) However, compounds represented by the following formulas are excluded:

[Chemical 11]

[Chemical 12] (13) The sodium salt of the above (12), wherein in the formula [III-1], L ⁴ is a methine group substituted with an alkyl group having 1 to 4 carbon atoms. (14) Sodium salt of the compound of formula [III-2] having 3 or more sulfonic acid groups in the molecule:

[Chemical 13] (In the formula, R ¹⁹ -R ²⁸ , X ′ and Y ′ have the same meanings as defined above; Z ³ is a non-metal atom group necessary for forming a 5- or 6-membered ring, and A is a hydrogen atom or The sodium salt of (12) above, which is a monovalent group. (15) The following formula:

[Chemical 14] The sodium salt of the above (14) represented by: (16) The following formula:

[Chemical 15] The sodium salt of (12) above represented by: (17) The sodium salt according to any one of (9), (10), (12), (13) and (14), which contains 4 or more sulfonic acid groups in the molecule. (18) The sodium salt of any of (9), (10), (12), (13), (14) and (17), which contains 10 or less sulfonic acid groups in the molecule. (19) The sodium salt according to any one of the above (9), (10), (12), (13), (14) and (17) containing 8 or less sulfonic acid groups in the molecule. (20) A near-infrared fluorescent contrast agent containing the sodium salt according to any one of (9) to (19). (21) The near-infrared fluorescent contrast agent according to (20) above, which is used for tumor imaging and / or angiography. (22) A fluorescence imaging method including the step of introducing the near-infrared fluorescent contrast agent according to (1) above into a living body, the step of irradiating the living body with excitation light, and the step of detecting near-infrared fluorescence from the above-mentioned contrast agent. . (23) At least one sodium salt of (9) above selected from the group consisting of compounds of the following formulas:

[Chemical 16]

[Chemical 17]

[Chemical 18] (24) At least one sodium salt of the above (12) selected from the group consisting of compounds of the following formulas:

[Chemical 19]

[Chemical 20]

[Chemical 21]

[Chemical formula 22]

[Chemical formula 23]

[Chemical formula 24]

[Chemical 25]

[Chemical formula 26] (25) The near-infrared fluorescent contrast agent of the above (1), which comprises at least one compound selected from the group consisting of compounds of the following formulas:

[Chemical 27]

[Chemical 28]

[Chemical 29]

[Chemical 30]

[Chemical 31]

[Chemical 32]

[Chemical 33]

[Chemical 34]

[Chemical 35]

[Chemical 36]

[Chemical 37]

[Chemical 38]

[Chemical Formula 39]

[Chemical 40] (26) The monovalent group A is a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aralkyl group, a lower alkoxy group, an optionally substituted substituted amino group, an alkylcarbonyloxy A group, a substituted or unsubstituted alkylthio group, a substituted or unsubstituted arylthio group,
A cyano group, a nitro group or a hydrogen atom, (14)
Sodium salt.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The terms used in the present specification are defined below. The near-infrared fluorescent contrast agent of the present invention means a contrast agent that emits fluorescence in the near-infrared region. The sulfonic acid group in the present invention, when the sulfonic acid group is used to form internal salts of the sulfonic acid salt (-SO ₃ ^-)
Can also mean. Preferred X and Y in the present invention are represented by the following formulas:

[Chemical 41] (In the formula, R ³ and R ⁴ are the same or different and each is a substituted or unsubstituted alkyl group).

The alkyl group of the "substituted or unsubstituted alkyl group" for R ¹ , R ² , R ³ and R ⁴ is preferably methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, sec. -Butyl group, tert-butyl group, pentyl group, isopentyl group, neopentyl group, tert-pentyl group, 2-methylpropyl group, 1,1-dimethylpropyl group, etc. having 1 to 5 carbon atoms A chain or branched lower alkyl group. The substituent may be, for example, a sulfonic acid group, a carboxyl group, a hydroxyl group, or the like. Examples of the substituted alkyl group include hydroxymethyl group, 1-hydroxyethyl group, 2-hydroxyethyl group, 2-hydroxypropyl group, 3-hydroxypropyl group, 4-hydroxybutyl group, carboxymethyl group, carboxyethyl group. , Carboxybutyl group, sulfomethyl group, 2-sulfoethyl group, 3-sulfopropyl group, 4-sulfobutyl group and the like. Preferred R ¹ and R ² are 1 substituted with a sulfonic acid group
To lower alkyl groups having from 5 to 5 carbon atoms (eg,
2-sulfoethyl group, 3-sulfopropyl group, 4-sulfobutyl group, etc., and R ³ and R ⁴ are unsubstituted lower alkyl groups having 1 to 5 carbon atoms (eg, methyl group, ethyl group, etc.) ).

Unsubstituted lower alkyl groups having 1 to 5 carbon atoms in R ¹⁷ and R ¹⁸ are, for example, R ¹ above,
"Substituted or unsubstituted alkyl group" for R ² , R ³ and R ⁴
The alkyl groups mentioned above can be mentioned. Examples of the alkyl group of the lower alkyl group having 1 to 5 carbon atoms which is substituted with the sulfonic acid group in R ¹⁹ and R ²⁰ include the above-mentioned “substituted or substituted” in R ¹ , R ² , R ³ and R ⁴ . Examples of the substituted lower alkyl group having 1 to 5 carbon atoms include 2-sulfoethyl group, 3-sulfopropyl group and 4 -Sulfobutyl groups.

The alkyl part of the alkyl (sulfoalkyl) amino group, bis (sulfoalkyl) amino group, sulfoalkoxy group, (sulfoalkyl) sulfonyl group and (sulfoalkyl) aminosulfonyl group in R ^{21 to} R ²⁸ is preferably 1 A straight-chain or branched lower alkyl group having from 5 to 5 carbon atoms, for example R ¹ , R ² , R ³ and R ^{4 above.}
Examples of the alkyl group of the “substituted or unsubstituted alkyl group” in are.

In the present invention, the "non-metal atom required to form a substituted or unsubstituted fused benzo ring or fused naphtho ring" means a bonding group required to form a fused benzo ring or fused naphtho ring. And is a group represented by the following formula:

[Chemical 42] When the condensed benzo ring or condensed naphtho ring has a substituent, the bonding group may include a substituent. Specific examples thereof include carbon atom, nitrogen atom, oxygen atom, hydrogen atom, sulfur atom, halogen atom (for example, fluorine atom,
Chlorine atom, bromine atom and iodine atom) and the like.

Examples of the substituents of the condensed benzo ring and condensed naphtho ring formed by the non-metal atom in Z ¹ and Z ² include sulfonic acid group, carboxyl group, hydroxyl group, halogen atom (for example, fluorine atom, chlorine atom). , Bromine atom and iodine atom), cyano group, substituted amino group (for example, dimethylamino group, diethylamino group, ethyl 4-sulfobutylamino group, di- (3-sulfopropyl) amino group, etc.), and
There are substituted or unsubstituted alkyl groups as defined above attached directly to the ring or by a divalent linking group.
Preferred divalent linking groups are, for example, -O-, -NHCO-, -NHSO.
_{2 -, - NHCOO -, -} NHCONH -, - COO -, - CO-, SO 2 - or the like. Examples of the alkyl group of the substituted or unsubstituted alkyl group which is bonded to the ring directly or by a divalent bonding group are preferably a methyl group, an ethyl group, a propyl group and a butyl group, and examples of the substituents Of these, a sulfonic acid group, a carboxyl group and a hydroxyl group are preferable.

Examples of the substituent of the methine group in L ¹ -L ⁷ include a substituted or unsubstituted alkyl group (defined above), a halogen atom (defined above), a substituted or unsubstituted aryl group, a lower alkoxy group and the like. is there. Examples of the aryl group of the “substituted or unsubstituted aryl group” include a phenyl group and a naphthyl group, but a phenyl group is preferable. Examples of the above substituent include a halogen atom (as defined above, preferably chlorine atom) and the like. Examples of the substituted aryl group include a 4-chlorophenyl group. The lower alkoxy group is preferably a linear or branched alkoxy group having 1 to 6 carbon atoms, especially methoxy group, ethoxy group, propoxy group, butoxy group, tert-
A butoxy group, a pentyloxy group and the like are preferable, and a methoxy group and an ethoxy group are preferable. Further, the substituents of the methine group in L ¹ -L ⁷ may combine with each other to form a ring containing three methine groups, and this ring may further form a condensed ring with a ring containing different methine groups. Good. An example of the ring containing three methine groups formed by the bonding of the substituents of the methine group in L ¹ -L ⁷ is 4,4-dimethylcyclohexene ring.

The conjugated methine chain having a ring of L ¹ -L ⁷ and having a ring is preferably a group of formula (a):

[Chemical 43] (In the formula, Z ³ represents a non-metal atom necessary to form a 5- or 6-membered ring, and A is a hydrogen atom or a monovalent group).

Examples of the "non-metal atom necessary for forming a 5- or 6-membered ring" include those mentioned above.
In the formula (a) and the formula [III-2] described below, examples of the 5- or 6-membered ring in Z ³ include a cyclopentene ring, a cyclohexene ring, a 4,4-dimethylcyclohexene ring, and the like. It is a cyclopentene ring.

As the monovalent group represented by A, for example,
Substituted or unsubstituted alkyl group (defined above), substituted or unsubstituted aryl group (defined above), substituted or unsubstituted aralkyl group, lower alkoxy group (defined above), optionally substituted substituted amino group, alkyl Examples thereof include a carbonyloxy group (eg, acetoxy group), a substituted or unsubstituted alkylthio group, a substituted or unsubstituted arylthio group, a cyano group, a nitro group, a halogen atom (as defined above) and the like. As used herein, examples of the aralkyl group of the “substituted or unsubstituted aralkyl group” include a benzyl group, a 2-phenylethyl group, a 1-phenylethyl group, a 3-phenylpropyl group, and the like. The group may be a sulfonic acid group, a carboxyl group, a hydroxyl group, a substituted or unsubstituted alkyl group (defined above), an alkoxy group (defined above), a halogen atom (defined above) and the like. "Optionally substituted substituted amino group"
Examples of the substituted amino group include, for example, an alkylamino group (eg, methylamino group, ethylamino group, etc.), dialkylamino group (dimethylamino group, diethylamino group, etc.), diphenylamino group, methylphenylamino group,
Examples thereof include a cyclic amino group (eg, morpholino group, imidazolidino group, ethoxycarbonylpiperazino group, etc.). Examples of the substituent relating to the arbitrary substitution of the “optionally substituted substituted amino group” include a sulfonic acid group and a carboxyl group. "Substituted or unsubstituted alkylthio group"
The alkylthio group may be, for example, a methylthio group or an ethylthio group. Examples of the substituent include a sulfonic acid group and a carboxyl group. Examples of the arylthio group of the “substituted or unsubstituted arylthio group” include a phenylthio group and a naphthylthio group. Examples of the substituent include a sulfonic acid group and a carboxyl group.

The monovalent group represented by A is preferably a fluorine atom, a chlorine atom or a dialkylamino group (preferably 6).
Or morpholino groups having up to 4 carbon atoms and optionally forming a ring. This group particularly preferably has a sulphonic acid group.

In the formula [I], r is preferably 1.
The pharmaceutically acceptable salt may be any salt as long as it forms a non-toxic salt with the compound of the formula [I]. Examples thereof include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as magnesium salt, calcium salt and the like; ammonium salt, triethylammonium salt, tributylammonium salt, pyridinium salt and the like. Organic ammonium salts; amino acid salts such as lysine salts, arginine salts and the like. Particularly preferred is sodium salt, which has low toxicity in vivo.

The fluorescent contrast agent to be used in vivo needs to be particularly water soluble. Water solubility of the near-infrared fluorescent contrast agent of the present invention is remarkably improved by introducing three or more sulfonic acid groups into the above compound. In order to obtain excellent water solubility, the number of sulfonic acid groups is preferably 4 or more. To facilitate the synthesis, the number of sulfonic acid groups is 10 or less, preferably 8 or less. The improvement in water solubility can be investigated by measuring the partition coefficient of each compound, for example by measuring the partition coefficient in a butanol / water two-phase system. More specifically, by introducing three or more sulfonic acid groups, the partition coefficient of n-butanol / water log Po / w
Is less than -1.00.

The sulphonic acid groups are R ¹ , R ² , Z ¹ and / or of the formula [I]
Alternatively, it is particularly preferable to introduce Z ² and the position of R ¹ , R ² , R ⁵ , R ⁷ , R ¹¹ and / or R ¹³ of the formula [II]. Further, these sulfonic acid groups are preferably introduced into L ⁴ of the conjugated methine chain at the position A of the above formula (a) via a divalent group such as an alkylene group.

Of the sodium salts of compounds of formula [II] having 3 or more sulphonic acid groups in the molecule, lower ones having 1 to 5 carbon atoms in which R ¹ and R ² are substituted by sulphonic acid groups. An alkyl group; X and Y are the same or different and each has the following formula

[Chemical 44] (Wherein R ¹⁷ and R ¹⁸ are the same or different and each is an unsubstituted lower alkyl group having 1 to 5 carbon atoms), the sodium salt of the compound is preferred, and the salt is And a compound having 3 or more sulfonic acid groups, particularly preferred are compounds of the following formulas.

[Chemical formula 45]

Among the compounds of formula [I] and pharmaceutically acceptable salts thereof having 3 or more sulphonic acid groups in the molecule, preferred are the sodium salts of compounds of formula [III-1]:

[Chemical formula 46] Where R ¹ -L ⁷ are as defined above; R ¹⁹ and R ²⁰ are lower alkyl groups having 1 to 5 carbon atoms substituted with sulfonic acid groups; R ²¹ to R ²⁸ Are the same or different and each represents a hydrogen atom, a sulfonic acid group, a carboxyl group, a hydroxyl group, an alkyl (sulfoalkyl) amino group, a bis group.
A (sulfoalkyl) amino group, a sulfoalkoxy group, a (sulfoalkyl) sulfonyl group or a (sulfoalkyl) amino-sulfonyl group; X ′ and Y ′ are the same or different and each has the following formula

[Chemical 47] (Wherein R ¹⁷ and R ¹⁸ have the same meanings as defined above)}, the salt has 3 or more sulfonic acid groups in the molecule, and particularly preferred are the compounds of the following formulas: is there.

[Chemical 48]

Among the sodium salts of the compound of formula [III-1] having 3 or more sulfonic acid groups in the molecule, preferred is the sodium salt of the compound of formula [III-2]:

[Chemical 49] (Wherein R ¹⁹ -R ²⁸ , X ′ and Y ′ have the same meanings as defined above; Z
³ is a non-metal atom necessary to form a 5- or 6-membered ring; A is a hydrogen atom or a monovalent group), the salt has 3 or more sulfonic acid groups in the molecule, Particularly preferred are compounds of the formula:

[Chemical 50]

The compound contained in the near-infrared fluorescent contrast agent of the present invention has 3 or more, preferably 4 or more sulfonic acid groups in the formula and is represented by the formula [I] or [II]. As long as it is anything. These compounds are "Cyanine dyes and related compounds", FM Hamer, John Wiley and So.
ns, New York, 1964; Cytometry, 10, 3-10 (1989); Cyt
ometry, 11, 418-430 (1990); Cytometry, 12, 723-730
(1990); Bioconjugate Chem. 4, 105-111 (1993), Ana
l. Biochem., 217, 197-204 (1994), Tetrahedron, 45,
4845-4866 (1989), EP-A-0591820A1, EP-A-0580145A1
It can be synthesized by a known method for producing a cyanine dye compound disclosed in et al. Alternatively, they can be semi-synthesized from commercially available cyanine dye compounds by known methods. In particular, they can be synthesized by reacting a dianyl compound and a quaternary salt of a heterocyclic compound.

The compound of the formula [I] of the present invention can be synthesized, for example, by the following method. (i) When r = 0, (a) L ¹ = L ⁵ , X = Y, R ¹ = R ² and Z ¹ = Z ² heterocyclic quaternary salt compound of the formula [IV-1] (2 mol)

[Chemical 51] (In the formula, L ¹ , X, Z ¹ and R ¹ are as defined above) and the dianyl compound of the formula [V-1] (1 mol)

[Chemical 52] (Wherein L ² , L ³ and L ⁴ are as defined above) in the presence of a base and a solvent to give a compound of formula [VI-1]

[Chemical 53] (Wherein L ¹ , L ² , L ³ , L ⁴ , R ¹ , Z ¹ and X have the same meanings as defined above), and this compound [VI-1] (1 mol) and necessary mol An amount of the compound of formula [VII] T ¹ -Na [VII] (wherein T ¹ is an organic acid residue) to react with the above formula [VI-
The sodium salt of the compound of 1] is obtained.

(B) L ¹ ≠ L ⁵ or X ≠ Y or R ¹ ≠ R ² or Z ¹ ≠ Z ² The heterocyclic quaternary salt compound (1 mol) of the above formula [IV-1] and the formula
A compound of the formula [VIII-1] is obtained by reacting the above-mentioned dianyl compound of [V-1] (1 mol) in the presence of a base and a solvent.

[Chemical 54] (Wherein L ¹ , L ² , L ³ , L ⁴ , R ¹ , Z ¹ and X have the same meanings as defined above), and this compound [VIII-1] (1 mol) and formula [XI -1] heterocyclic quaternary salt compound (1 mol)

[Chemical 55] (Wherein L ⁵ , Y, Z ² and R ² have the same meanings as defined above), and then the compound of formula [X-1]

[Chemical 56] (Wherein, L ¹ , L ² , L ³ , L ⁴ , L ⁵ , R ¹ , R ² , Z ¹ , Z ² , X and Y
Is the same as the above definition), and the compound of the formula [X-1] (1 mol) is reacted with a necessary molar amount of the compound of the formula [VII] to give the above formula [X-1]. A sodium salt of the compound of

(Ii) When r = 1, (a) L ¹ = L ⁷ , X = Y, R ¹ = R ² and Z ¹ = Z ² a heterocyclic tetrasalt compound of the formula [IV-1] ( 2 mol)

[Chemical 57] (In the formula, L ¹ , X, Z ¹ and R ¹ are as defined above) and the dianyl compound of the formula [V-2] (1 mol)

[Chemical 58] (Wherein L ² , L ³ , L ⁴ , L ⁵ and L ⁶ have the same meanings as defined above) and are reacted in the presence of a base and a solvent to give a compound of formula [VI-2]

[Chemical 59] (Wherein L ¹ , L ² , L ³ , L ⁴ , L ⁵ , L ⁶ , R ¹ , Z ¹ and X have the same meanings as defined above), and the compound [VI-2] (1 compound T ¹ -Na [VII] (wherein the mole) and a necessary molar amount of a compound of formula [VII], T ¹ is by reacting the definition is as defined above) the formula
The sodium salt of the compound of [VI-2] is obtained.

(B) L ¹ ≠ L ⁷ or X ≠ Y or R ¹ ≠ R ² or Z ¹ ≠ Z ² The heterocyclic quaternary salt compound (1 mol) of the above formula [IV-1] and the formula
A compound of the formula [VIII-2] is obtained by reacting the above-mentioned dianyl compound of [V-2] (1 mol) in the presence of a base and a solvent.

[Chemical 60] (Wherein L ¹ , L ² , L ³ , L ⁴ , L ⁵ , L ⁶ , R ¹ , Z ¹ and X have the same meanings as defined above), and the compound [VIII-2] (1 And a heterocyclic tetrasalt compound of the formula [IX-2] (1 mol)

[Chemical formula 61] (Wherein L ⁷ , Y, Z ² and R ² have the same meanings as defined above), and then the compound of formula [X-2]

[Chemical formula 62] (Wherein, L ¹ , L ² , L ³ , L ⁴ , L ⁵ , L ⁶ , L ⁷ , R ¹ , R ² , Z ¹ ,
Z ² , X and Y have the same meanings as defined above), and the formula [X
-2] (1 mol) and the required molar amount of the above compound of formula [VII] are reacted to obtain the sodium salt of the above compound of formula [X-2].

(Iii) When r = 2, when r is 2, L ⁶ and L ⁷ are duplicated in the formula [I]. To avoid this, overlapping L ⁶ and L ⁷ will be referred to as L ⁸ and L ⁹ for purposes of illustration. (a) L ¹ = L ⁹ , X = Y, R ¹ = R ² and Z ¹ = Z ² A heterocyclic quaternary salt compound of the formula [IV-1] (2 mol)

[Chemical formula 63] (In the formula, L ¹ , X, Z ¹ and R ¹ have the same meanings as defined above) and the dianyl compound of the formula [V-3] (1 mol).

[Chemical 64] (Wherein L ² , L ³ , L ⁴ , L ⁵ , L ⁶ and L ⁷ have the same meanings as defined above, and L ⁸ is an optionally substituted methine group) in the presence of a base and a solvent. Reacted compound of formula [VI-3]

[Chemical 65] (Wherein, L ¹ , L ² , L ³ , L ⁴ , L ⁵ , L ⁶ , L ⁷ , L ⁸ , R ¹ , Z ¹ and
X has the same meaning as defined above) to obtain this compound [VI-3]
(1 mole) and compound T ¹ -Na [VII] (wherein, T ¹ is as defined above and is synonymous) of the formula [VII] in the required molar amount above formula by reacting
A sodium salt of the compound of [VI-3] is obtained.

(B) L ¹ ≠ L ⁹ or X ≠ Y or R ¹ ≠ R ² or Z ¹ ≠ Z ² The heterocyclic quaternary salt compound (1 mol) of the above formula [IV-1] and the formula
A compound of the formula [VIII-3] is obtained by reacting the above-mentioned dianyl compound of [V-3] (1 mol) in the presence of a base and a solvent.

[Chemical formula 66] (Wherein, L ¹ , L ² , L ³ , L ⁴ , L ⁵ , L ⁶ , L ⁷ , L ⁸ , R ¹ , Z ¹ and
X is as defined above) to obtain this compound [VIII-
3] (1 mol) and a heterocyclic quaternary salt compound of the formula [IX-3] (1
Mol)

[Chemical formula 67] (Wherein Y, Z ² and R ² have the same meanings as defined above, and L ⁹ is an optionally substituted methine group) to react with the compound of formula [X-3]
Compound of

[Chemical 68] (Wherein, L ¹ , L ² , L ³ , L ⁴ , L ⁵ , L ⁶ , L ⁷ , L ⁸ , L ⁹ , R ¹ ,
R ² , Z ¹ , Z ² , X and Y are as defined above, and
The compound of the formula [X-3] (1 mol) and the necessary molar amount of the formula
The sodium salt of the compound of the above formula [X-3] is obtained by reacting the above compound of [VII].

The necessary molar amount of the compound of the formula [VII] is an amount equal to or more than the amount of sodium contained in 1 mol of the intended sodium salt of the compound of the formula [I]. L ⁸ and
Examples of the substituent of the substituted methine group in L ⁹ include:
There is a description about the substituent of the above methine group in L ¹ to L ⁷ .

In the above synthetic methods (i), (ii) and (iii), the reaction of the compounds [IV-1] and [V-1], the reaction of the compounds [VIII-1] and [XI-1], Reaction of compounds [IV-1] and [V-2], reaction of compounds [VIII-2] and [IX-2], compounds [IV-1] and [V-3]
And the reactions of compounds [VIII-3] and [IX-3] are -20
It is carried out at a temperature of -80 ° C, preferably -10-40 ° C, preferably in the presence of an acylating agent such as acetic anhydride.

In the above synthetic methods (i), (ii) and (iii), the reaction of the compounds [IV-1] and [VII], the compounds [X-1] and [V]
II] reaction, compound [VI-2] and [VII] reaction, compound [X-
2] and [VII], the compounds [VI-3] and [VII], and the compounds [X-3] and [VII] are preferably 0-40
It is carried out at a temperature of ° C, preferably in the presence of a solvent such as alcohol or water.

In the above synthetic methods (i), (ii) and (iii), the base to be used is, for example, triethylamine,
It may be tributylamine, pyridine, diazabicycloundecene, sodium methoxide, etc .; the solvent to be used is, for example, N, N-dimethylacetamide, N-methylpyrrolidone and N, N-diethylformamide or methanol. Alcohols may be used; organic acid residues are eg CH ₃ C
It may be OO or the like.

With respect to the preparation of various pharmaceutically acceptable salts of the compound of the above formula [I], the ammonium salt and potassium salt of the compound of the above formula [I] can be prepared by, for example, the above synthetic methods (i) and (ii).
And (iii) can be obtained by replacing the compound of formula [VII] with a compound of formula [VII] in which a sodium atom is changed to an ammonium group or a potassium atom; Different cation salts of the compound can be obtained by converting the ammonium salt and potassium salt into different cation salts, if necessary, using an ion exchange resin.

Specific examples of the compound of the above formula [I] including the compound of the formula [II] used in the present invention are shown below, but the present invention is not limited thereto.

[Chemical 69]

[Chemical 70]

[Chemical 71]

[Chemical 72]

[Chemical formula 73]

[Chemical 74]

[Chemical 75]

[Chemical 76]

[Chemical 77]

[Chemical 78]

[Chemical 79]

[Chemical 80]

[Chemical 81]

The above compound to be contained in the near-infrared fluorescent contrast agent of the present invention exhibits absorption and fluorescence in the near-infrared light region of 700-1300 nm, particularly about 700-900 nm, and has a molar extinction coefficient of 100,000 or more. Have.

The near infrared fluorescent contrast agent of the present invention has the formula [I] or the formula
It is not particularly limited as long as it contains the compound [II] and / or a pharmaceutically acceptable salt thereof and has 3 or more, preferably 4 or more sulfonic acid groups in the molecule. This compound or a salt thereof may be contained in the contrast medium alone or in combination.

Specifically, the contrast agent contains the above compound or the above compound suspended or dissolved in a solvent such as distilled water for injection, physiological saline, Ringer's solution and the like. If necessary, pharmacologically acceptable additives such as carriers and excipients may be added. Examples of these additives include pharmacologically acceptable substances such as electrolytes, buffers and detergents, and substances for adjusting osmotic pressure to improve stability and solubility (for example, cyclodextrin, liposomes, etc.). . A wide variety of additives commonly used in the relevant fields can be used. The near-infrared fluorescent contrast agent of the present invention is preferably manufactured through sterilization for pharmaceutical use. The contrast agent is
It can be administered to a living body by injection, spraying or application, intravascular (venous, arterial), oral, intraperitoneal, transdermal, subcutaneous, intravesical or intrabronchial administration. Preferably, the contrast agent is administered intravascularly in the form of a 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 can finally detect the site to be diagnosed, and the compound that emits near-infrared fluorescence is used. It is appropriately adjusted depending on the type, age, body weight, target organ to be administered, and the like. Typically, the dose is 0.1-100 mg / kg body weight, preferably 0.5, as the amount of the compound.
-20 mg / kg body weight. The contrast agent of the present invention can be appropriately used for various animals other than humans. The form, route and dose of administration are appropriately determined depending on the weight and condition of the subject animal.

In the present invention, further, 3 or more in the molecule,
The compounds of the formula [I], which preferably have 4 or more sulphonic acid groups, particularly preferably the compounds of the formula [II], tend to be significantly accumulated in the tumor tissue. By utilizing this characteristic, tumor tissue can be specifically imaged using the fluorescent contrast agent of the present invention. Furthermore, a series of such compounds can stay in the blood vessel for a long time and are expected to function well as angiographic agents.

The fluorescence imaging method of the present invention is characterized by the use of the near infrared fluorescent contrast agent of the present invention. This method is performed according to known methods, and the respective parameters such as the excitation wavelength and the fluorescence wavelength to be detected enable optimal imaging and evaluation depending on the type of the near-infrared fluorescent contrast agent to be administered and the administration target. It is determined as appropriate. The time taken from the administration of the near-infrared fluorescent contrast agent of the present invention to the start of measurement by the fluorescence imaging method of the present invention varies depending on the type of the near-infrared fluorescent contrast agent used and the administration target. For example, if the contrast agent comprises a compound of formula [I] for tumor imaging purposes, the elimination time will be approximately 4-120 hours after administration. In the case of the compound of formula [II],
The elimination time is considered to be approximately 24-120 hours after administration. If the disappearance time is too short, the fluorescence will be too strong to clearly separate the target site from other sites. If it is too long, the contrast agent may be excreted from the body. When angiography is desired, the compound of formula [I] or formula [II] is detected immediately after administration or within about 30 minutes thereof.

The method typically includes the following steps. That is, the near-infrared fluorescent contrast agent of the present invention is administered to a detection target, and the detection target is exposed to excitation light from an excitation light source. Then, the fluorescence generated by the excitation light from the near infrared fluorescent contrast agent is detected by the fluorescence detector. The excitation wavelength depends on the near-infrared fluorescent contrast agent used, and is not limited as long as the compound emits fluorescence in the near-infrared region effectively. Preferably, near-infrared light having excellent bio-penetrating ability is used.

The wavelength of near infrared fluorescence to be detected also depends on the contrast agent used. Generally speaking, 600-1000 n
Near-infrared fluorescence in the wavelength range of 700-1000 nm, preferably 750-900 nm is detected using excitation light having a wavelength of m, preferably 700-850 nm. In this case, the excitation light source may be an ordinary excitation light source such as various lasers (eg, ion laser, dye laser, and semiconductor laser), halogen light source, xenon light source, and the like. If desired, various optical filters can be used to obtain the optimum excitation wavelength. Similarly, fluorescence can be detected by capturing only fluorescence from the near-infrared fluorescent contrast agent using various optical filters.

The detected fluorescence is data-processed as fluorescence information and used to generate a recordable fluorescence image. The fluorescence image is generated by illuminating a wide area including the target tissue, detecting the fluorescence with a CCD camera, and image-processing the obtained fluorescence information. Or optical CT
The device may be used, an endoscope may be used, and a fundus camera may be used. The fluorescence imaging method of the present invention can visualize systemic diseases, tumors, blood vessels, etc. without damaging the living body.

The present invention will be described in more detail by way of examples and experimental examples, but the present invention is not limited thereto. The compound numbers in the following Examples and Experimental Examples correspond to the compound numbers explained by the structural formulas. Compounds in which a symbol representing "potassium salt", "calcium salt" or "pyridinium salt" is shown after the compound number (eg:
Compound (29) K salt) means the same compound as the compound (sodium salt) represented by the compound number except that the counter ion is potassium salt, calcium salt or pyridinium salt instead of sodium salt. . For example, `` compound (3
"1) K salt" means the same compound as compound (31), except that the counterion is potassium instead of sodium;
"Compound (31) Ca salt" means the same compound as compound (31) except that the counterion is calcium instead of sodium; "Compound (31) pyridinium salt" is the counterion instead of sodium. It means the same compound as the compound (31) except that it is pyridinium. A method for synthesizing a compound contained in the near-infrared fluorescent contrast agent of the present invention as an active ingredient will be described in Examples.

The following synthetic methods mostly consist of the reaction of the heterocyclic quaternary salt compounds shown in Table 1 with the dianyl compounds shown in Tables 2 and 3.

[Table 1]

[Table 2]

[Table 3]

[0051]

EXAMPLES In the following examples, the compounds are represented by the symbols used in Tables 1 to 3 (eg, A1, Q1, etc.) for convenience. Example 1: Synthesis of compound (29) Heterocyclic quaternary salt compound Q1 (5 g) was added to methanol (100 m
l), N, N-dimethylformamide (25 ml), triethylamine (5.6 ml), dianil compound A1 (1.83 g) and acetic anhydride (3 ml) were added, and the mixture was stirred at room temperature for 4 hours. Triethylamine (2.2 ml) and acetic anhydride (2 ml) were added, and the mixture was stirred at room temperature for 3 hours. Insoluble material was removed by filtration, and sodium acetate (2 g) in methanol (15 ml) was added.
The solution was added to the filtrate and stirred at room temperature for 1 hour. The formed crystals were collected by filtration and washed with a small amount of methanol. Water (20 ml) was added to the obtained crude crystal (3.5 g) to dissolve it. After adding sodium acetate (1 g), methanol (30 ml) was added and stirred for 1 hour. The resulting crystals are collected by filtration,
After washing with a small amount of methanol and drying, 3 g of the compound (2
Got 9). The obtained compound (29) showed a yellow flame reaction. Maximum absorption wavelength (H ₂ O): 780 nm Molar extinction coefficient (H ₂ O): 243,000 Maximum fluorescence emission wavelength (H ₂ O): 802 nm About the obtained compound (29), the infrared absorption spectrum of the compound (29) is transformed by Fourier transform infrared It was measured by the potassium bromide tablet method using a meter (VALOR-III, manufactured by JASCO). The following peaks were detected. The spectrum is shown in FIG. IR (<max (KBr)): 1414, 1086, 1037, 995, 889 cm ^-1

Example 2: Synthesis of compound (34) A heterocyclic quaternary salt compound Q2 (2.13 g) was added to methanol (20 m).
l) was added and the mixture was cooled to 10 ° C. Dianyl compound A2 (0.75 g), triethylamine (4 ml) and acetic anhydride (2 ml) were added thereto, and the mixture was stirred for 20 minutes. Acetic anhydride (2 ml) was added and the mixture was stirred at 10 ° C for 4 hours. The insoluble material was removed by filtration, and a solution of sodium acetate (2 g) in a small amount of methanol was added to the filtrate. The obtained crystals were collected by filtration and washed with a small amount of methanol.
Water (7 ml) was added to the obtained crude crystals to dissolve them. Methanol (7 ml) was added to precipitate crystals. The crystals were collected by filtration, washed with a small amount of methanol, dried, and 1.
2 g of compound (34) was obtained. The obtained compound (34) showed a yellow flame reaction. Maximum absorption wavelength (H ₂ O): 794 nm Molar extinction coefficient (H ₂ O): 176,000 Maximum fluorescence emission wavelength (H ₂ O): 812 nm

Example 3: Synthesis of compound (6) Heterocyclic quaternary salt compound Q3 (9.5 g) was added to methanol (50
ml), triethylamine (7 ml), dianil compound A3
(3.1 g) and acetic anhydride (3.9 ml) are added and the mixture is stirred at room temperature for 7 hours. Insoluble matter was removed by filtration, and a solution of sodium acetate (5 g) in a small amount of methanol was added to the filtrate. Leave the mixture overnight. The crystals obtained are collected by filtration and washed with a little methanol. Water (30 ml) is added to the crystals to dissolve them. Sodium acetate (2 g) was added, followed by methanol (30 ml). The obtained crystals are collected by filtration, washed with a small amount of methanol and dried to obtain compound (6).

Example 4: Synthesis of compound (45) A heterocyclic quaternary salt compound Q3 (4.8 g) was added to methanol (50 m
l), triethylamine (4 ml), dianil compound A4 (1.7
g) and acetic anhydride (2 ml) were added, and the mixture was stirred at room temperature for 3 hours. Insoluble matter is removed by filtration, sodium acetate
A solution of (4 g) in a small amount of methanol was added to the filtrate. The obtained crystals were collected by filtration and washed with a small amount of methanol. Water (10 ml) was added to the crystals to dissolve them. Then methanol (10 ml) was added. The obtained crystals were collected by filtration, washed with a small amount of methanol and dried, except that the substituent on the methine chain was -Cl instead of -SCH ₂ CH ₂ SO ₃ Na. 1.6 g of the same compound as). The above steps were repeated to obtain 4.2 g of the compound. Water (30 ml), triethylamine (1.2 ml) and sodium 2
-Mercaptoethane sulfonate (0.8 g) was added and the mixture was stirred at room temperature for 4 hours. The insoluble material was removed by filtration, and a solution of sodium acetate (2 g) in a small amount of water was added to the filtrate. The obtained crystals were collected by filtration, washed with methanol (20 ml), and air-dried to obtain 2.3 g of compound (45). The obtained compound (45) showed a yellow flame reaction. Maximum absorption wavelength (H ₂ O): 815 nm Molar extinction coefficient (H ₂ O): 196,000 Maximum fluorescence emission wavelength (H ₂ O): 827 nm

Example 5: Synthesis of compound (2) Heterocyclic quaternary salt compound Q3 (4.7 g) was added to methanol (25 m
l), triethylamine (2.8 ml), dianil compound A5
(1.5 g) and acetic anhydride (2.4 ml) are added and the mixture is stirred at room temperature for 1 hour. Triethylamine there
(3.5 ml) and acetic anhydride (1.5 ml) are added and the mixture is stirred at room temperature for 3.5 hours. Insoluble matter was removed by filtration, and a solution of sodium acetate (3 g) in a small amount of methanol was added to the filtrate. The mixture is stirred at room temperature for 1 hour. The crystals obtained are collected by filtration and washed with a little methanol. Water (15 ml) is added to the crystals to dissolve them. Then methanol (15 ml) is added. The crystals obtained are collected by filtration, washed with a small amount of methanol and dried to give the compound
Get (2).

Example 6: Synthesis of compound (43) Heterocyclic quaternary salt compound Q3 (3.75 g) was added to methanol (25 m
l), triethylamine (3.5 ml), dianil compound A6
(1.95 g) and acetic anhydride (2.4 ml) were added, and the mixture was stirred at room temperature for 1 hr. The insoluble matter was removed by filtration, and a solution of sodium acetate (3.9 g) in a small amount of methanol was added to the filtrate. The mixture was stirred at room temperature for 1 hour.
The obtained crystals were collected by filtration and washed with a small amount of methanol. Water (10 ml) was added to the crystals to dissolve them. Sodium acetate (2 g) was added, followed by methanol (10 ml). The obtained crystals were collected by filtration, washed with a small amount of methanol and dried to obtain 1.8 g of compound (43). The obtained compound (43) showed a yellow flame reaction. Maximum absorption wavelength (H ₂ O): 773 nm Molar extinction coefficient (H ₂ O): 204,000 Maximum fluorescence emission wavelength (H ₂ O): 789 nm

Example 7: Synthesis of compound (4) A heterocyclic quaternary salt compound Q3 (3.5 g) was added to methanol (20 m
l), triethylamine (3.5 ml), dianil compound A7
(1.2 g) and acetic anhydride (1.9 ml) are added, the mixture is stirred at room temperature for 10 hours and then left overnight. This mixture
Stir for 5 hours while heating to 50 ° C. Add water (2 ml),
Insoluble matter is removed by filtration. A solution of sodium acetate (5 g) in a small amount of water is added to the filtrate. The mixture at room temperature
Stir for 30 minutes. The obtained crystals are collected by filtration, washed with a small amount of methanol and dried to obtain compound (4).

Example 8: Synthesis of compound (31) A heterocyclic quaternary salt compound Q4 (3.5 g) was added to methanol (35 m
l), triethylamine (3.5 ml) and acetic anhydride (2 ml) were added, and the dianyl compound A2 (1.8 g) was added in several batches with stirring. The mixture was stirred for another hour.
Acetic anhydride (2 ml) was added and the mixture was stirred at room temperature for 5 hours. Insoluble material was filtered off and sodium acetate (4 g)
Was dissolved in a small amount of methanol and added to the filtrate. The obtained crystals were collected by filtration and washed with a small amount of methanol. Water (10 ml) was added to the crystals to dissolve them. Methanol (10 ml) was then added and the mixture was stirred at room temperature for 2 hours. The obtained crystals were collected by filtration, washed with a small amount of methanol and dried to obtain 1.3 g of compound (31). The obtained compound (31) showed a yellow flame reaction. Maximum absorption wavelength (H ₂ O): 755 nm Molar extinction coefficient (H ₂ O): 228,000 Maximum fluorescence emission wavelength (H ₂ O): 774 nm Infrared absorption spectrum of the obtained compound (31) is calculated by Fourier transform infrared spectroscopy. It was measured by the potassium bromide tablet method using a meter (VALOR-III, manufactured by JASCO). The following peaks were detected. The spectrum is shown in FIG. IR (νmax (KBr)): 1518, 1183, 1149, 1111, 995 cm ^-1

Example 9: Synthesis of compound (41) Heterocyclic quaternary salt compound Q1 (12 g) was added to methanol (120 m
l), triethylamine (13.6 ml), dianil compound A8
(4.4 g) and acetic anhydride (2.4 ml) were added and the mixture was added to 30
Stir for minutes. Acetic anhydride (2.4 ml) was added and the mixture was mixed with 1.5
After stirring for an hour, acetic anhydride (2.4 ml) was further added, and the mixture was stirred at room temperature for 6 hours. Heterocyclic quaternary salt compound Q1 (1
g), triethylamine (3 ml) and acetic anhydride (3 ml) were further added, and the mixture was stirred at room temperature for 2 hours and left overnight. Sodium acetate (5 g) was added, and the formed crystals were collected by filtration and washed with a small amount of methanol. Water (200 ml) was added to the obtained crude crystals. Insoluble material was removed by filtration, and sodium acetate (10 g) was added to the filtrate. The obtained crystals were collected by filtration and washed with a small amount of methanol. Water (200 ml) and triethylamine (10 ml) were added to the crystals,
A solution of sodium acetate (10 g) in methanol (100 ml) was added to obtain crystals. This process was repeated twice. The obtained crystals were collected by filtration, washed with a small amount of methanol and dried to obtain 9.7 g of compound (41). The obtained compound (41) showed a yellow flame reaction. Maximum absorption wavelength (H ₂ O): 811 nm Molar extinction coefficient (H ₂ O): 230,000 Maximum fluorescence emission wavelength (H ₂ O): 822 nm

Example 10: Synthesis of compound (3) According to Example 5, compound (3) is obtained using heterocyclic quaternary salt compound Q3 and the corresponding dianyl compound.

Example 11 By a method similar to the synthesis of compound (29) in Example 1, except that potassium acetate (2 g) was used in place of sodium acetate (2 g), the counter ion was sodium. The same compound as compound (29) was obtained except that it was potassium instead. Hereinafter, this compound is referred to as Compound (29) K salt. The obtained compound (29) K salt showed a purple flame reaction. Maximum absorption wavelength (H ₂ O): 780 nm Molar extinction coefficient (H ₂ O): 254,000 Maximum fluorescence emission wavelength (H ₂ O): 800 nm

The other compounds described above are treated in the same manner as in this example to obtain a compound having potassium as a counter ion instead of sodium. These compounds having potassium as a counterion are distinguished from the above compounds by writing "K salt" after the corresponding compound number.

Example 12 In the same manner as in Example 11, the compound (6) K salt was obtained. The obtained compound (6) K salt exhibited a purple flame reaction. Maximum absorption wavelength (H ₂ O): 788 nm Molar extinction coefficient (H ₂ O): 226,000 Maximum fluorescence emission wavelength (H ₂ O): 806 nm

Example 13 In the same manner as in Example 11, compound (2) K salt was obtained. The obtained compound (2) K salt exhibited a purple flame reaction. Maximum absorption wavelength (H ₂ O): 743 nm Molar extinction coefficient (H ₂ O): 266,000 Maximum fluorescence emission wavelength (H ₂ O): 762 nm

Example 14 In the same manner as in Example 11, the compound (4) K salt was obtained. The obtained compound (4) K salt exhibited a purple flame reaction. Maximum absorption wavelength (H ₂ O): 753 nm Molar extinction coefficient (H ₂ O): 212,000 Maximum fluorescence emission wavelength (H ₂ O): 767 nm

Example 15 In the same manner as in Example 11, compound (3) K salt was obtained. The obtained compound (3) K salt exhibited a purple flame reaction. Maximum absorption wavelength (H ₂ O): 751 nm Molar extinction coefficient (H ₂ O): 241,000 Maximum fluorescence emission wavelength (H ₂ O): 767 nm

Example 16 The compound (6) K salt (50 mg) was dissolved in a small amount of water, and potassium of the compound (6) K salt was converted to a proton through an ion exchange resin. Methanol saturated with sodium acetate was added thereto to precipitate crystals. This process was repeated twice.
The obtained crystals were collected by filtration, washed with a small amount of methanol and dried to obtain 32 mg of compound (6). The obtained compound (6) showed a yellow flame reaction. Obtained compound (6)
The infrared absorption spectrum was measured by a potassium bromide tablet method using a Fourier transform infrared spectrometer (VALOR-III, manufactured by JASCO). The following peaks were detected. The spectrum is shown in FIG. IR (νmax (KBr)): 1395, 1372, 1188, 1102, 1020 cm ^-1

Example 17: Synthesis of compound (54) Heterocyclic quaternary salt compound Q4 (3.5 g) was added to methanol (20 m
l), triethylamine (3.5 ml) and acetic anhydride (2 ml) were added, and the dianyl compound A1 (1.4 g) was added in several batches with stirring. The mixture was stirred for another 20 minutes.
Acetic anhydride (1 ml) was added and the mixture was stirred at room temperature for 1.5 hours. The insoluble material was removed by filtration, and a solution of sodium acetate (4 g) in a small amount of methanol was added to the filtrate. The obtained crystals were collected by filtration and washed with a small amount of methanol. The crystals were dissolved in a small amount of water. The solution was then diluted with methanol (10 ml) and the mixture was stirred at room temperature for 1 hour. The obtained crystals were collected by filtration, washed with a small amount of methanol and dried to obtain 1.5 g of compound (54). The obtained compound (54) showed a yellow flame reaction. Maximum absorption wavelength (H ₂ O): 743 nm Molar extinction coefficient (H ₂ O): 244,000 Maximum fluorescence emission wavelength (H ₂ O): 766 nm Infrared absorption spectrum of the obtained compound (54) is calculated by Fourier transform infrared spectroscopy. It was measured by the potassium bromide tablet method using a meter (VALOR-III, manufactured by JASCO). The following peaks were detected. The spectrum is shown in FIG. IR (νmax (KBr)): 1511, 1421, 1099, 1004, 926 cm ^-1

Experimental Example 1 The partition coefficient (log Po / w) of n-butanol / water was calculated from compound (29),
Compound (43), compound (45), compound (31), compound (3) K salt,
Compound (11) [obtained from Japan Photosensitive Dye Research Institute Co., Ltd. as NK-3261], Compound (6) K salt, Compound (2) K salt, Compound (4) K salt,
It measured about the compound (34) and the compound (54). As a control compound, NK-19, which has only two sulfonic acid groups in the molecule
67 (Japan Photosensitive Dye Research Institute) and ICG (Tokyo Kasei) were used. The results are shown in Table 4.

[Table 4]

Experimental Example 2: Fluorescence Imaging Test (1) Tumor tissue fragments of mouse colon cancer (26 cancers of the colon) were subcutaneously transplanted into the left chest of BALB / c nude mice (5 weeks old, Claire Japan). After 10 days, when the tumor had grown to a diameter of about 8 mm, the mice were tested. A titanium sapphire laser was used as the fluorescence excitation light source. Irradiation dispersion is 10
The test mouse was uniformly irradiated with laser light using a ring type light guide (Sumitomo Optical Glass Co., Ltd.) so that the ratio was within%. Irradiation power is about 40 μW near the skin surface of the mouse
It was adjusted to be / cm ² . Fluorescence was excited at the maximum excitation wavelength of each compound, and the fluorescence emission from the mouse was cut by using a CCD camera (C4880, Hamamatsu Photonics KK) with short wavelength cut-off filters (IR84, IR86, IR88; Fuji Photo Film).
Detected and photographed. The cutoff filter was chosen to match the excitation wavelength of the compound. The irradiation time was adjusted by the fluorescence intensity of each compound.

The test compounds used were the compounds of the invention (2
9), Compound (31) and Compound (6) K salt and NK-1967 and IC having only two sulfonic acid groups in the molecule as control compounds
G. Each test compound was dissolved in distilled water (0.5 mg / m
l), the mice were administered via the tail vein. Dose is compound (3
1), Compound (6) K salt, NK-1967 and ICG 5.0 mg /
kg, 0.5 mg / kg for compound (29). Compound administration 2
After 4 hours, the mouse was anesthetized with diethyl ether, and a fluorescence image of the whole mouse was photographed. The results are shown in Figures 1 to 5.

The compound (29) having a benzotricarbocyanine structure and 6 sulfonic acid groups is a compound (6) K having both a tricarbocyanine structure and 4 sulfonic acid groups.
Similar to salt and compound (31), a reference compound with two sulfonic acid groups (having a benzotricarbocyanine structure
Compared to NK-1967 and ICG with tricarbocyanine structure), it clearly produced sharper images. In particular, the compound (29) could clearly depict tumor even at a low dose, and had a remarkable effect.

Experimental Example 3: Fluorescence imaging test (2) Nude mice were used for the test. The compound (29) of the present invention and the control compound ICG were intravenously administered via the caudal vein at a dose of 5.0 mg / kg under continuous inhalation anesthesia with sevoflurane. At the same time, we started to take fluorescent images intermittently. In capturing a fluorescence image, exposure to an excitation laser beam and extraction of fluorescence through a filter were performed, and the exposure time was 1 second.
Twenty seconds after compound administration, blood vessels were properly imaged. Fluorescent images were taken up to 5 minutes after administration. 6 to 9 show fluorescence images of the whole mouse 20 seconds and 5 minutes after the administration. Although ICG did not show blood vessels by contrast at 5 minutes, compound (29) was able to image blood vessels for a longer time than ICG.

Experimental Example 4: Retention in Blood Vessel In the same manner as in Experimental Example 2, a tumor tissue fragment was transplanted into a CDF ₁ mouse (female, 5 weeks old, Japan SLC), and after about 2 weeks, the diameter of the tumor was increased. Mice were tested when they grew to approximately 1 cm. The test compound is a compound (29) K salt and compound (41) K salt having a benzotricarbocyanine structure and 6 sulfonic acid groups; a compound (6) having a tricarbocyanine structure and 4-5 sulfonic acid groups (6 ) K salt, compound (4) K salt, compound
(45) K salt, compound (31), compound (31) K salt, compound (3) K salt,
Compound (2) K salt, compound (43) K salt and compound (11); and control compounds ICG and NK-1967. Each test compound was dissolved in distilled water (0.5 mg / ml) and used. Each of the resulting compound solutions was administered via the tail vein of mice (5.0 mg / kg). Blood was collected from the mice 0.5, 1, 4 and 24 hours after compound administration and centrifuged to obtain plasma. The fluorescence intensity of the plasma was measured with a spectrofluorometer (RF 5300 PC, Shimadzu Corporation). A calibration curve for each compound was drawn and the compound concentration in plasma was calculated. The results are shown in Fig. 10. The compound of the present invention remained in plasma for a long time.

Experimental Example 5: Acute toxicity A reduction in toxicity due to the introduction of a sulfonic acid group and a reduction in toxicity due to conversion to a sodium salt were tested. The test compounds are shown in Table 5. Each test compound was dissolved in distilled water to prepare a compound solution. This solution was intravenously injected into awake mice through the tail vein. The mice were observed for 3 days after the administration to evaluate acute toxicity [LD ₅₀ (mg / kg body weight)]. The results are shown in Table 5.

[Table 5] Increasing the number of sulfonic acid groups in the molecule or conversion to the sodium salt significantly reduced acute toxicity.

The near-infrared fluorescent contrast agent of the present invention is excited by excitation light and emits near-infrared fluorescence. This infrared fluorescence is excellent in transmitting biological tissue. Therefore, it becomes possible to detect a lesion in a deep part of a living body. Furthermore, since the contrast agent of the present invention is excellent in water solubility and low toxicity, it can be safely used.

[Brief description of drawings]

FIG. 1 is a photograph showing fluorescence imaging 24 hours after administration of a compound. A: ICG (5 mg / kg) was administered.

FIG. 2 is a photograph showing fluorescence imaging 24 hours after the administration of the compound. B: NK-1967 (5 mg / kg) was administered.

FIG. 3 is a photograph showing fluorescence imaging 24 hours after the administration of the compound. C: Compound (29) (0.5 mg / kg) was administered.

FIG. 4 is a photograph showing fluorescence imaging 24 hours after the administration of the compound. D: Potassium salt of compound (6) (5 mg / kg)
Was administered.

FIG. 5 is a photograph showing fluorescence imaging 24 hours after the administration of the compound. E: Compound (31) (5 mg / kg) was administered.

FIG. 6 is photographs showing fluorescence imaging 20 seconds and 5 minutes after administration of the compound (5 mg / kg). A: ICG (after 20 seconds)
Was administered.

FIG. 7 is photographs showing fluorescence imaging 20 seconds and 5 minutes after administration of the compound (5 mg / kg). B: ICG (after 5 minutes) was administered.

FIG. 8 is photographs showing fluorescence imaging 20 seconds and 5 minutes after administration of the compound (5 mg / kg). C: Compound (29)
(After 20 seconds) was administered.

FIG. 9 is photographs showing fluorescence imaging 20 seconds and 5 minutes after administration of the compound (5 mg / kg). D: Compound (29)
(After 5 minutes) was administered.

FIG. 10 is a graph showing the compound concentration in plasma 0.5, 1, 4 and 24 hours after administration of the compound. The vertical axis represents the concentration of the compound in plasma (μg / ml) at each time point.

FIG. 11 is a chart showing an infrared absorption spectrum of compound (29).

FIG. 12 is a chart showing an infrared absorption spectrum of compound (31).

FIG. 13 is a chart showing an infrared absorption spectrum of compound (6).

FIG. 14 is a chart showing an infrared absorption spectrum of compound (54).

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Riku Inagaki             3-16-16 Minoh, Minoh, Minoh City, Osaka Prefecture (72) Inventor Hiroaki Eguchi             2-72 Seiwadai Nishi, Kawanishi City, Hyogo Prefecture (72) Inventor Masafumi Okumura             1-21 Senno 1-chome, Otsu City, Shiga Prefecture (72) Inventor Yoshio Inagaki             Fuji Photo, 210 Nakanuma, Minamiashigara City, Kanagawa Prefecture             Within Film Co., Ltd. (72) Inventor Toru Harada             Fuji Photo, 210 Nakanuma, Minamiashigara City, Kanagawa Prefecture             Within Film Co., Ltd. F-term (reference) 4C085 HH01 KB56 LL01 LL18                 4C204 BB09 BB10 CB03 DB03 DB11                       DB12 DB13 EB10 FB15 GB29

Claims (4)

[Claims] 1. The following formula: The sodium salt represented by. 2. A near-infrared fluorescent contrast agent containing the sodium salt according to claim 1. 3. The near-infrared fluorescent contrast agent according to claim 2 for tumor imaging. 4. The near-infrared fluorescent contrast agent according to claim 2 for angiography.