JPH04288022A - Intracorporeal diagnosticum and treating agent for cancer or tumor, containing dyestuff for fluorescent labeling - Google Patents

Abstract

PURPOSE:To obtain a diagnosticum for cancer and tumor, having high accuracy and efficiency, containing a dyestuff for fluorescent labeling of phthalocyanine further condensed with 4 benzene rings, not being influenced by various spectrum disturbing factors. CONSTITUTION:A dyestuff for fluorescent labeling shown by the formula {M is H2, Al, Si, P, Ga, Ge, Cd, Sc, Mg, Sn or Zn; R<1> to R<4> are XQW, QW, W or H [X is O, N, S, P, Si, Se or CR<5>R<6> (R<5> and R<6> are H, alkyl, aryl, etc.) or phenylene; Q is bond group; W is OH, O<->, SH, S<->, CO2H, CO2<->, PO3<->, SO3<->, NH2, etc.]; k, l, m and n are 0-4; Y is halogen, OR<13> or NR2<14> (R<13> and R<14> are H, alkyl, acyl, silyl or P-containing group); p is 0-2; Z<1> to Z<8> are CH or N}, such as sodium sulfonate of chloroaluminum phthalocyanine. The dyestuff is bonded to a substance derived from an organism to be selectively linked to a tumor cell and used. An inexpensive small-sized semiconductor layer can be used.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an in-vivo diagnostic and therapeutic agent for cancer or tumor containing a fluorescent labeling dye.

0002

BACKGROUND OF THE INVENTION Phthalocyanine pigments are organic pigments having four isoindole moieties linked by four nitrogen atoms constituting a cyclic conjugated chain. Compounds used in these pigments include phthalocyanine (blue-green), copper phthalocyanine (blue), chlorine-substituted copper phthalocyanine (green), and sulfonated copper phthalocyanine (green). Phthalocyanine pigments are commonly used in enamel, plastics, linoleum, inks, wallpaper, textiles, paper, rubber products, etc. On the other hand, it has been reported that free-based phthalocyanine and phthalocyanine of aluminum, cadmium, magnesium, silicon, tin or zinc exhibit fluorescence (The Phtha
locyanines 1:127, 1983).

[0003] It has also been reported that phthalocyanine can be used in various immunoassays (US Pat. No. 4,
No. 160,645, US Pat. No. 4,193,983
US Patent No. 4,220,450, US Patent No. 4,233,402, US Patent No. 4,235
, No. 869, US Patent No. 4,256,834, US Patent No. 4,277,437, US Patent No. 4
, 318,707, US Pat. No. 4,483,92
No. 9, US Patent No. 4,540,660, US
Patent No. 4,540,670, US Patent No. 4,56
No. 0,534, US Patent No. 4,650,770, US Patent No. 4,656,129, US Patent No. 4,659,676).

Furthermore, phthalocyanines are used as catalysts in chemiluminescence immunoassay systems [Bull. Chem
．． Soc. Jpn. Volume 56, pages 2965-2968 (
1983), Vol. 56, pp. 2267-2271 (19
83), Vol. 57, pp. 587-588 (1984),
57th volume, pages 3009-3010 (1984), 58th volume, pages 1299-1303 (1985)]. Hara et al. use iron phthalocyanine as a catalyst for the chemiluminescent reaction between luminol and hydrogen peroxide, and quantify the analyte in the test sample from the amount of chemiluminescent signal. They contain iron and cobalt phthalocyanines as well as iron,
We investigated porphyrin complexes of palladium, platinum, manganese, and tin, and reported that iron phthalocyanine showed the best catalytic activity and high sensitivity.

On the other hand, it has been suggested that phthalocyanine can also be used in cancer radiotherapy (PDT) using a radiation light source (visible light) as a photosensitive reagent (sensitizer). To be useful in the treatment of cancer, sensitizers must first be delivered selectively to tumor cells. For example, monoclonal antibody-hematoporphyrin conjugates are available to deliver sensitizers to tumor cells (J. Immu.
nol. Vol. 130, pp. 1473-1477 (1983
)). Next, the sensitizer is excited with visible light to cause a photoreaction that involves the generation of singlet oxygen and energy transfer, killing the cells. In this case, tetrasulfonated aluminum and zinc phthalocyanine are said to be particularly effective for PDT [J. Radiat. Biol. Vol. 47, pp. 145-147 (1985), Photochem
．． and Photobiol. Volume 42 129-1
33 (1985), Radiat. Res. 103rd
Vol., pp. 403-409 (1985), Photoche
m. and Photobiol. Volume 42, 515
-521 pages (1985), Lasers in t
he Life Sciences Volume 1, 79-
86 (1986), Photochem. and
Photobiol. Volume 43, pp. 615-621 (1
986), Br. J. Cancer Volume 53, 255-
263 (1986), Racliat. Res. Vol. 107, pp. 136-142 (1986), J. Ur
ology Vol. 136, pp. 141-145 (198
6), Int. J. Radiat. Biol No. 51
Volume 467-476 (1987)].

[0006] Fluorescent substances are widely used in addition to colored substances in immunoassays, and fluorescent substances are also preferred over colored substances in enzyme immunoassays because they can increase sensitivity. It has become to. A well-known fluorescent substance-enzyme pair is alkaline phosphatase (al
kaline phosphatase) and 4-methylumbelliferyl phosphate (4-methyl
β-galactosidase and 4-methylumbelliferyl-D-galactopyranoside (4-methylumbelliferyl-D-g
horseradish peroxidase (alactopyranoside), horseradish peroxidase
roxidase) and p-hydroxyphenylacetic acid (p
-hydroxyphenyl acetic a
cid), etc., and the detection sensitivity of these systems is 10-15
It is M. However, since there are limits to the analytical properties of the produced fluorophore, it is difficult to further increase the detection sensitivity.

[0007]Recently, reagents using phthalocyanine that have a high fluorescence quantum yield and high solubility in water have been proposed (WO Patent No. 88/04777, WO Patent No. 9).
0/02747, Japanese Unexamined Patent Publication No. 1-233222).

[0008]

[Problems to be Solved by the Invention] However, phthalocyanine has a Q-band absorption range and a fluorescence emission range of 650 to 70.
Since it is in the 0 nm region and overlaps with the absorption range (<700 nm) of heme in blood, which is one of the substances in living organisms, there is a drawback that the fluorescent light emitted from phthalocyanine is absorbed by heme in blood.

In addition, it is thought that cheap and small semiconductor lasers (670 to 780 nm) will become mainstream in the future as radiation light sources, but when excited with a 670 to 690 nm semiconductor laser, phthalocyanine has a fluorescence emission region similar to this. Not only is it difficult to distinguish between scattered light from the irradiated laser light and fluorescent light emission because of the wavelength range of 700~7
When excited with an 80 nm semiconductor laser, phthalocyanine is not excited because it cannot absorb light, and therefore does not serve as a detection agent.

[0010] The present invention is not affected by biological substances such as heme present in the blood, and can also be performed using an inexpensive and small semiconductor laser (670 to 780 nm), which is expected to become mainstream in the future. The purpose is to provide in-vivo diagnostic and therapeutic agents for tumors.

[0011]

[Means for Solving the Problems] The present invention provides the following (1) to (
2). That is, (1) chemical formula 3

[C3
] [In chemical formula 3, M is H2, Al, Si, P, Ga, Ge
, Cd, Sc, Mg, Sn or Zn, R1 , R
2 , R3 and R4 are each independently -XQW
, -QW, -W or a hydrogen atom, X is an oxygen atom,
Nitrogen atom, sulfur atom, phosphorus atom, silicon atom, selenium atom, CR5 R6 (However, R5 and R6 are each independently a hydrogen atom, an alkyl group, an aryl group, or an aralkyl group, and R5 R6 may be carbonyl oxygen. ), or a phenylene group, Q represents a bonding group (linker) between X and W, and W is -OH, -O -
, -SH, -S - , -CO2 H, -CO2 -
, -OCH2 CO2 H, -OCH2 CO2 -
, -PO42 - , -PO3- , -SO3-, -
SO2- , -SO2 Cl, -SO4 2 - , -
NH2 , -NHR7 , -NR8 R9 or -N(
+) R10R11R12 (However, R7 to R12 are
each independently a C1 to C10 alkyl group, a C6 to C10 alkyl group;
It is a C12 aryl group or a C6 to C12 aralkyl group. ), k, l, m and n each independently represent an integer of 0 to 4, Y is a halogen atom, -OR1
3 or -NR142 (However, R13 and R14 are
Each independently a hydrogen atom, an alkyl group that may have a hydrophilic substituent, an acyl group that may have a hydrophilic substituent, and a silyl group that may have a hydrophilic substituent. or a phosphorus atom-containing group which may have a hydrophilic substituent. ), and p is
Indicates an integer from 0 to 2 representing the number of bonds of Y to M, Z1
~Z8 each independently represent a methylene group or a nitrogen atom. ] An in-vivo diagnostic agent for detecting cancer or tumor containing a fluorescent labeling dye. (2) A therapeutic agent for cancer or tumor containing a fluorescent labeling dye represented by chemical formula 3.

In the compound of formula 3 in the present invention, R
Specific examples of the alkyl group for 5 and R6 include methyl group, ethyl group, propyl group, sec-propyl group, n-
There are butyl groups, iso-butyl groups, t-butyl groups, pentyl groups, hexyl groups, etc. Examples of aryl groups are phenyl groups, thienyl groups, furyl groups, pyrrolyl groups, tolyl groups, anisyl groups, 4-amino There are phenyl groups, etc., and aralkyl groups include benzyl group, 2-phenylethyl group, 1-phenylethyl group, 3-phenylpropyl group, 2-phenylethyl group, etc.
-phenylpropyl group, 1-phenylpropyl group, etc. Further, specific examples of the alkyl group that may have a hydrophilic substituent for R13 and R14 include a methyl group,
Ethyl group, propyl group, butyl group, pentyl group, hexyl group, heptyl group, octyl group, nonyl group, decyl group,
There are straight chain, branched and alicyclic groups such as dodecyl group, tetradecyl group, hexadecyl group, octadecyl group, eicosyl group, and docosyl group, and examples of acyl groups that may have hydrophilic substituents include , formyl group, acetyl group, propionyl group, butyryl group, valeryl group, pivaloyl group, hexanoyl group, octanoyl group, lauryl group, palmityl group, stearyl group, and silyl group which may have a hydrophilic substituent. The groups include trimethylsilyl group, triethylsilyl group, tripropylsilyl group, tributylsilyl group, triamylsilyl group, trihexylsilyl group, t-butyldimethylsilyl group, di(
Examples include t-butyl)methylsilyl group, dimethylphenylsilyl group, diphenylmethylsilyl group, and triphenylsilyl group.

Q is a group that connects X and W, and is a C1 to C8 saturated or unsaturated linear, branched or alicyclic bonding group;
For example, in addition to methylene group, ethylene group, trimethylene group, tetramethylene group, propylene group, vinylene group, propenylene group, cyclopropylene group, cyclopentylene group, cyclohexylene group, etc., groups such as polyether, polyamine, polyalcohol, etc. There is.

In W, the C1 to C10 alkyl groups of R7 to R12 include methyl group, ethyl group, propyl group,
There are linear, branched, and cyclic groups such as butyl group, amyl group, hexyl group, heptyl group, nonyl group, and decyl group, and C
Examples of the 6-C12 aryl group include phenyl group, tolyl group, anisyl group, naphthyl group, biphenyl group, etc.
C6 to C12 aralkyl groups include benzyl group, 2
-phenylethyl group, 1-phenylethyl group, 3-phenylpropyl group, 2-phenylpropyl group, 1-phenylpropyl group, etc.

In addition, the substituents containing a phosphorus atom represented by R13 and R14 include (R15 to R19 each independently represent an alkyl group, an aryl group, an acyl group, a cycloalkyl group, an alkoxyl group, an aryloxyl group) , a polyether group, a hydroxyl group, or a halogen atom, which may have various substituents).

In formula 3, Y is -OR13 or -NR142
(R13 and R14 represent an alkyl group, an acyl group, and a silyl group), and the compound in which p represents 1 or 2 is:
A compound in which Y is -OH or -NH2 in Chemical Formula 3 can be synthesized by reacting with a corresponding alcohol, acyl chloride, silanol, chlorosilane, chlorophosphine, chlorophosphite, phosphoryl chloride, or the like. In Chemical Formula 3, Y is -OH or -NH2, and p is 1 or 2. In Chemical Formula 3, Y is a halogen atom,
It can be obtained by hydrolyzing or ammonolyzing a compound in which p is 1 or 2. During 3rd year,
Compounds in which Y is a halogen atom and p is 1 or 2, and compounds in which p is zero and do not have Y can be synthesized by the following two routes.

The first route is described in the literature (Zh. Obshch.
Khim Vol. 39, pp. 2554-2558 (1969
2) By the method described, Chemical 4

[Formula 4] (wherein, M is H2, Al, Si, P, Ga, Ge, C
d, Sc, Mg, Sn or Zn, Y represents a halogen atom, p represents an integer of 0 to 2 representing the number of bonds of Y to M, and Z1 to Z8 each independently represent a methylene group or a nitrogen atom. shows. ) is obtained, and then R1 , R2 , R3 or R4 which can form a substituent R1 , R2 , R3 or R4 in Chemical Formula 3 is obtained.
This is a method for obtaining a compound represented by chemical formula 3 by reacting a chemical species having a moiety.

The second route is chemical formula 5

[C5] Or 6

[Chemical Formula 6] (In Chemical Formula 5 and Chemical Formula 6, Z1 , Z2 , R1 and k are
It can be obtained by reacting a compound represented by the formula (same meaning as in Chemical Formula 3) with a corresponding metal or metal salt. On the other hand, when M in Chemical Formula 4 is H2, it can be obtained by reacting the compounds represented by Chemical Formulas 5 and 6 with Na alkoxide or Li alkoxide, and then hydrolyzing. The compound represented by Chemical Formula 6 can be obtained by reacting the compound represented by Chemical Formula 5 with ammonia in methanol in the presence of sodium methoxide.

In the present invention, in order for the fluorescent labeling dye represented by Chemical Formula 3 to be applied to an in-vivo diagnostic and therapeutic agent for cancer or tumor cells, the fluorescent labeling dye represented by Chemical Formula 3 must be applied to cancer or tumor cells. It must be delivered selectively to tumor cells. For that purpose, biological substances that selectively bind to cancer or tumor cells may be used. Specific examples include antiserum obtained from the blood of animals immunized by sensitizing cancer or tumor cells, such as mice, rabbits, sheep, horses, and cows, polyclonal antibodies isolated and purified from the antiserum, Furthermore, there are monoclonal antibodies prepared using hybridoma technology.

[0020] In order to bond the fluorescent labeling dye to the above-mentioned biological substance, it is necessary to combine the functional groups such as amino groups and hydroxyl groups in the biological substance with the carboxyl group and sulfone group in the fluorescent labeling dye. The biological material is either directly bonded ionic or covalently, or chemically modified by adding a linker to a part of the biological material so that it can react with a fluorescent labeling dye. You can react later. A biological substance labeled with a fluorescent labeling dye can be purified by conventional separation means such as chromatography and ammonium sulfate precipitation.

The fluorescent labeling dye represented by Chemical Formula 3 or the biological substance labeled with it is dissolved in an aqueous solution or physiological saline together with pharmaceutically acceptable stabilizers, excipients, etc. and administered into the body. After a certain period of time, the cancer or tumor tissue is taken up by cancer or tumor cells, and then the cancer or tumor tissue is detected by irradiating it with light and measuring the generated fluorescence. When the detected site is irradiated with laser light, the fluorescent labeling dye taken up by cancer or tumor cells is excited, and the ensuing dark reaction generates oxygen active species (singlet oxygen or superoxide anion radicals). , resulting in the destruction of cancer or tumor cells.

[0022] An important point for an in-vivo diagnostic agent that detects cancer or tumors using fluorescence is that the substance has a high fluorescence quantum yield.
, Al, Si, P, Ga, Ge, Cd, Sc, Mg,
Sn and Zn are used. In addition, in order to achieve a high fluorescence quantum yield, the fluorescent labeling dye in the present invention must be
It is necessary to bind to cancer or tumor cells in a single molecule state without intermolecular association. Y coordinates with M, and in vivo, M is further coordinated with water molecules, -SH groups, -NH2 groups, etc. in proteins, resulting in steric hindrance, which inhibits intermolecular association, resulting in high It is believed that a fluorescence quantum yield is achieved.

On the other hand, when used as a therapeutic agent for cancer and tumors, in order to be effective, the fluorescent labeling dye must have excellent light absorption ability as a sensitizer. The fluorescent labeling dye in the present invention exhibits a maximum absorption at a wavelength of 700 nm or more in a single molecule state [
Since the molar absorption coefficient ε at Q(0,0) is 300,000 to 700,000, it is important to maintain a monomolecular state. Furthermore, the fluorescent labeling dye in the present invention exhibits high solubility in water,
After being taken into the living body, it has a high fluorescence quantum yield and a sensitizing effect, making it an excellent in-vivo diagnostic and therapeutic agent for cancer or tumors.

The compound represented by Chemical Formula 3 has four benzene rings fused to phthalocyanine, so its absorption and fluorescence wavelength range is longer than that of phthalocyanine (
700 nm or more), it is immune to the effects of various spectral interference factors in living organisms, and therefore the fluorescent labeling dye represented by chemical formula 3 or biological substances labeled with it has high precision and efficiency. It can be used as a good in-vivo diagnostic and therapeutic agent for cancer or tumors.

[0025]

EXAMPLES The present invention will be explained in more detail below using synthesis examples and test examples. Synthesis Example 1 Chloroaluminum naphthalocyanine sulfonic acid sodium salt literature (Zh. Obshch. Khim. Vol. 39, 25
Chloroaluminum naphthalocyanine (1.5 g) synthesized by the method described on pages 54-2558 (1969)
Add 5 ml of chlorosulfonic acid and incubate at 65°C under nitrogen for 6 hours.
Stir for hours. After cooling, the contents were poured into 100 g of ice. The obtained solid was filtered, washed with water and then with methanol, and then dried under reduced pressure. To the obtained solid (200 mg),
A 1N-NaOH aqueous solution (1 ml) was added, and the mixture was stirred at room temperature for 24 hours. After stirring, the reaction mixture was purified by chromatography method (10 wt% NH4OH/methanol (weight ratio: 1/
3)), 0.32 g of a dark green solid was obtained. Analysis of this solid revealed that about three sulfonic acid groups had been introduced. The absorption and fluorescence emission spectra of this compound in an aqueous solution are shown in FIG.

Synthesis Example 2 Zinc naphthalocyanine sulfonic acid sodium salt literature (Zh. Obshch. Khim. Vol. 39, 25
Zinc naphthalocyanine (1.5 g) synthesized by the method described on pages 54-2558 (1969) was treated in the same manner as in Synthesis Example 1 to obtain 0.33 g of a dark green solid. Analysis of this solid revealed that about three sulfonic acid groups had been introduced.

Synthesis Example 3 Sulfonic acid sodium salt of dihydroxysilicon naphthalocyanine Literature (J. Am. Chem. Soc. Vol. 106, 74)
Dihydroxysilicon naphthalocyanine (1.5 g) synthesized by the method described on page 04-7410 (1984)
) was treated in the same manner as in Synthesis Example 1 to obtain 0.31 g of a black-green solid. Analysis of this solid revealed that about three sulfonic acid groups had been introduced.

Synthesis Example 4 Sodium sulfonate salt of dibutoxysilicon naphthalocyanine The sodium sulfonate salt of dihydroxysilicon naphthalocyanine obtained in Synthesis Example 3 (1 g) and n-butanol (1 ml) were added to 30 ml of quinoline. Refluxed for an hour. After cooling, the reaction mixture was filtered and washed with toluene. The obtained solid was purified by chromatography to obtain 0.4 g of a green solid.

Synthesis Example 5 Sulfonic acid sodium salt of chloraluminum quinoxalocyanine literature (Zh. Obshch. Khim. Vol. 39, 25
Chloroaluminiumquinoxalocyanine (1.5
g) in the same manner as in Synthesis Example 1 and the literature (US Pat. No. 4,657,554) to obtain a black-green solid of 0.3
5g was obtained. Analysis of this solid revealed that about three sulfonic acid groups had been introduced.

Synthesis Example 6 Zinc quinoxalocyanine sulfonic acid sodium salt Literature (Zh. Obshch. Khim. Vol. 39, 25
Zinc quinoxalocyanine (1.5 g) obtained by the method described on pages 36-2541 (1969) was treated in the same manner as in Synthesis Example 1 and the literature (US Pat. No. 4,657,554) to obtain 0 black-green solids. .32g was obtained. Analysis of this solid revealed that about three sulfonic acid groups had been introduced. The absorption and fluorescence emission spectra of this compound in an aqueous solution are shown in FIG.

Synthesis Example 7 Sulfonic acid sodium salt of chloroaluminum quinocyanine Literature (Zh. Obshch. Khim. Vol. 39, 25
Chloroaluminum quinocyanine (1.5 g) synthesized according to the method described on pages 36-2541 (1969)
was treated in the same manner as in Synthesis Example 1 and the literature (US Pat. No. 4,657,554) to obtain 0.35 g of a black-green solid.
was gotten. Analysis of this solid revealed that about three sulfonic acid groups had been introduced. The absorption and fluorescence emission spectra of this compound in an aqueous solution are shown in FIG.

Synthesis Example 8 Zinc quinolocyanine sulfonic acid sodium salt literature (Zh. Obshch. Khim. Vol. 39, 25
Zinc quinolocyanine (1.5 g) synthesized according to the method described on pages 36-2541 (1969) was treated in the same manner as in Synthesis Example 1 and the literature (US Pat. No. 4,657,554) to obtain 0.35 g of a black-green solid. was gotten. Analysis of this solid revealed that about three sulfonic acid groups had been introduced.

Synthesis Example 9 Tetracarboxylic acid sodium salt of aluminum naphthalocyanine Synthesized according to the method described in US Pat. No. 4,833,264. 1% by weight of aluminum tetrakis(n-amyloxycarbonyl)naphthalocyanine (100mg)
Reflux in a mixture of NaOH (1.2 ml) and ethanol (1 ml) at about 120°C for 3 hours, and after cooling, the pH of the solution
After confirming that the mixture was neutral, it was concentrated under reduced pressure. The obtained solid was transferred to a Soxhlet extractor, extracted with toluene for 6 hours, and then extracted with ethanol/water (2/1 by weight).
) The mixture was extracted for 5 hours to obtain a solution containing a water-soluble aluminum naphthalocyanine tetracarboxylic acid sodium salt. This was purified by chromatography to obtain aluminum naphthalocyanine tetracarboxylic acid sodium salt.

Synthesis Example 10 Tetracarboxylic acid sodium salt of zinc naphthalocyanine Amyl tetracarboxylate (2) of zinc naphthalocyanine synthesized by the method described in US Pat. No. 4,833,264
00mg) in the same manner as in Synthesis Example 9,
0.13 g of a dark green solid was obtained. The absorption and fluorescence emission spectra of this compound in an aqueous solution are shown in FIG.

Synthesis Example 11 Tetracarboxylic acid sodium salt of chloroaluminum quinoxalocyanine 6-amyloxycarbonyl-2,3-dicyanoquinoxaline (1
．． 4g), aluminum chloride (210mg), ammonium molybdate (10mg), and urea (5g) were heated at about 220°C for 2.5 hours while stirring well. After cooling, 40 ml of methanol was added to the solidified reaction mixture, and the mixture was thoroughly stirred at 50° C. for 30 minutes. Insoluble materials were collected by filtration and washed with methanol and then with acetone. The obtained solid was transferred to a Soxhlet extractor and extracted with methanol for 100 hours and then with acetone for 50 hours to remove impurities. Next, change the extraction solvent to chloroform and
Time Soxhlet extraction. The resulting dark green chloroform solution was filtered while hot, and the filtrate was concentrated and dried under reduced pressure to obtain 210 mg of black solid. By treating this solid in the same manner as in Synthesis Example 9,
．． 15 g of a dark green solid was obtained. FIG. 5 shows the absorption and fluorescence emission spectra of this compound in an aqueous solution.

Synthesis Example 12 Tetracarboxylic acid sodium salt of zinc quinoxalocyanine 6-amyloxycarbonyl-2,3-dicyanoquinoxaline (1.5 g), zinc powder (105 mg), ammonium molybdate (10 mg) and Urea (5 g) was treated in the same manner as in Synthesis Example 11 to obtain 800 mg of a dark green solid.

Synthesis Example 13 Sodium sulfonate salt of bis(trimethylsiloxy)silicon naphthalocyanine The sodium sulfonate salt of dihydroxysilicon naphthalocyanine obtained in Synthesis Example 3 (1 g) and trimethylsilanol (2 ml) were added to 30 ml of quinoline. ,2
Refluxed for an hour. After cooling, the reaction mixture was filtered and washed with toluene. The obtained solid was purified by chromatography to obtain 0.3 g of a green solid. Figure 6 shows the absorption and fluorescence emission spectra of this compound in an aqueous solution.
Shown below.

Synthesis Example 14 Sulfonic acid sodium salt of bis(triethylsiloxy)silicon naphthalocyanine [0038] The procedure was repeated in the same manner as in Synthesis Example 13, except that triethylsilanol was used instead of trimethylsilanol. Sulfonic acid sodium salt was obtained.

Synthesis Example 15 Aluminum naphthalocyanine mono{N-(p-hydroxycarbonylphenyl)
Sodium sulfamoyl}disulfonate Aluminum naphthalocyanine trisulfonic acid (150 mg) obtained in Synthesis Example 1 before converting into sodium salt with NaOH aqueous solution
) was added dropwise to 2 ml of benzene solution at 25°C with oxalyl chloride (0.75 ml). After stirring at room temperature for 6 hours, the solvent was removed to obtain aluminum naphthalocyanine trisulfonyl chloride as a dark green solid. On the other hand, p-aminobenzoic acid (PABA) (31 m
g) was added. After stirring at 80° C. for 5 minutes, aluminum naphthalocyanine trisulfonyl chloride (55 mg) was added. The reaction mixture was stirred at 80° C. for 6 hours and the solvent was removed. The resulting solid was diluted with methanol containing 10 wt% NH4OH, concentrated again, and ground with acetone to form aluminum naphthalocyanine mono{N-(p-
Sodium hydroxycarbonylphenyl)sulfamoyl}disulfonate was obtained.

Synthesis Example 16 Zinc naphthalocyanine trisulfonic acid (40 m
To a solution of g) in benzene (2 ml) was added dropwise oxalyl chloride (0.75 ml) at 25°C. After stirring at room temperature for 6 hours, the solvent was removed to obtain zinc naphthalocyanine trisulfonyl chloride as a dark green solid. On the other hand, in a solution of Na2CO3 (24 mg) in water (1 ml),
At 80°C, 2,2'-oxybis(ethylamine) hydrochloride (15mg) was added. After stirring at 80° C. for 5 minutes, the zinc naphthalocyanine trisulfonyl chloride prepared previously was added. Add another 0.5 ml of the reaction mixture
When diluted with water and heated at 80℃ for 12 hours, zinc naphthalocyanine mono[2-(2'-aminoethoxy)
Ethylaminosulfonyl disulfonic acid sodium salt was obtained.

Synthesis Example 17 Oxalyl chloride (0.75 ml) was added to a 2 ml benzene solution of aluminum quinoxalocyanine trisulfonic acid (150 mg) obtained in Synthesis Example 5 before converting it into a sodium salt with an aqueous NaOH solution at 25°C. dripped. At room temperature 6
After stirring for an hour, the solvent was removed to obtain aluminum quinoxalocyanine trisulfonyl chloride as a dark green solid. Meanwhile, PABA (31 mg) was added to a solution of Na2CO3 (61 mg) in water (1 ml) at 80°C. After stirring at 80° C. for 5 minutes, aluminum quinoxalocyanine trisulfonyl chloride (55 mg) was added. The reaction mixture was stirred at 80° C. for 6 hours and the solvent was removed. The obtained solid was diluted with methanol containing 10% by weight of NH4OH, concentrated again, and ground with acetone to obtain aluminum quinoxalocyanine mono-PABA-sulfonyldisulfonic acid sodium salt.

Synthesis Example 18 Zinc quinoxalocyanine trisulfonic acid (40
mg) in benzene (2 ml), oxalyl chloride (0.75 ml) was added dropwise at 25°C. After stirring at room temperature for 6 hours, the solvent was removed to obtain zinc quinoxalocyanine trisulfonyl chloride as a dark green solid. Meanwhile, 2,2'-oxybis(ethylamine) was added to a solution of Na2CO3 (24 mg) in water (1 ml) at 80°C.
Hydrochloride (15 mg) was added. After stirring at 80° C. for 5 minutes, the previously prepared zinc quinoxalocyanine trisulfonyl chloride was added. Add another 0.5 to the reaction mixture.
When diluted with ml of water and heated at 80°C for 12 hours,
Zinc quinoxalocyanine mono[2-(2'-aminoethoxy)ethylaminosulfonyl disulfonic acid sodium salt was obtained.

Synthesis Example 19 Zinc quinolocyanine trisulfonic acid (150 m
To 2 ml of benzene solution of g), oxalyl chloride (0.75 ml) was added dropwise at 25°C. After stirring at room temperature for 6 hours, the solvent was removed to obtain zinc quinocyanine trisulfonyl chloride as a dark green solid. on the other hand,
To a solution of Na2CO3 (61 mg) in water (1 ml), 80
PABA (31 mg) was added at °C. After stirring at 80°C for 5 minutes, zinc quinolocyanine trisulfonyl chloride (5
5 mg) was added. The reaction mixture was stirred at 80° C. for 6 hours and the solvent was removed. The obtained solid was mixed with 10% by weight of NH4
After diluting with methanol containing OH, concentrating again and grinding with acetone, zinc naphthalocyanine mono-PAB
A-sulfonyldisulfonic acid sodium salt was obtained.

Synthesis Example 20 Aluminum quinocyanine trisulfonic acid (obtained in Synthesis Example 7) before converting it into a sodium salt with an aqueous NaOH solution (
Oxalyl chloride (0.75 ml) was added dropwise to a solution of 40 mg) in benzene (2 ml) at 25°C. At room temperature 6
After stirring for an hour, the solvent was removed to obtain aluminum quinocyanine trisulfonyl chloride as a dark green solid. Meanwhile, Na2CO3 (24 mg) in water (1
ml) solution at 80°C was added 2,2'-oxybis(ethylamine) hydrochloride (15mg). 80
After stirring at °C for 5 minutes, the aluminum quinocyanine trisulfonyl chloride prepared previously was added. The reaction mixture was further diluted with 0.5 ml of water and heated at 80°C for 12 hours, resulting in aluminum quinocyanine mono[2-(
2'-Aminoethoxy)ethylaminosulfonyl]disulfonic acid sodium salt was obtained.

Synthesis Example 21 In Synthesis Example 13, benzene (2 m
l) Add oxalyl chloride (0.75 m
l) was added dropwise. After stirring at room temperature for 6 hours, the solvent was removed to obtain bis(trimethylsiloxy)silicon naphthalocyanine trisulfonyl chloride as a green solid. Meanwhile, Na2CO3 (24 mg) in water (1 ml)
) 2,2'-oxybis(ethylamine) hydrochloride (15 mg) was added to the solution at 80°C. After stirring at 80° C. for 5 minutes, the previously prepared bis(trimethylsiloxy)silicon naphthalocyanine trisulfonyl chloride was added. The reaction mixture was further diluted with 0.5 ml of water,
When heated at 80°C for 12 hours, bis(trimethylsiloxy)silicon naphthalocyanine mono[2-(2'aminoethoxy)ethylaminosulfonyl]disulfonic acid sodium salt was obtained.

Test Example 1 Toxicity test literature of fluorescent labeling dyes on cells [Photochem. Photobiol. Fourth
2, Vol. 515-521 (1985)], the effect of fluorescent labeling dyes on the cell viability of Chinese hamster lung fibroblasts V-79 was determined. Table 1 shows the results. From the results in Table 1, it can be seen that all fluorescent labeling compounds have a cell-killing effect.

[Table 1] ──────────────────────────
──────────── Compound

Cell death rate (%)──────
──────────────────────────
────── Sodium chloraluminum naphthalocyanine trisulfonic acid 70

Sodium zinc naphthalocyanine trisulfonate
66 Sodium dihydroxysilicon naphthalocyanine trisulfonate

75 Bis(trimethylsiloxy) silicon naphthalocyanine
Sodium trisulfonate
95
Sodium chloroaluminum quinoxalocyanine trisulfonate

80 Sodium zinc quinocyanine trisulfonate
85 Chloroaluminum naphthalocyanine tetracarboxylic acid 82 Sodium zinc quinoxalone anine tetracarboxylate
78 ──────────────────
────────────────────

0047
Test Example 2 Cancer Cell Detection Test After dissolving various fluorescent labeling dyes in physiological saline to a concentration of 10-5 mol/l, 0.5 ml of the solution was added to mice with peritoneal cancer (C3H/L).
MH134) was injected into the abdominal cavity with a syringe, and one hour later, 1 ml of ascites containing cancer cells was collected from the peritoneal cavity with a syringe. Add 4.5 ml of physiological saline to this 0.5 ml and
After centrifugation at 0 rpm for 5 minutes, the supernatant was removed, and the same operation was repeated twice to obtain peritoneal cancer cells. Take these peritoneal cancer cells into a syringe and hematocrit tube, and
After centrifugation at 000 rpm, a semiconductor laser (67
The fluorescence emission spectrum was observed using 0 nm). The results obtained are shown in Table 2. As is clear from the results in Table 2, cancer cells could be well detected using the compound of the present invention, but the fluorescence emission spectrum could not be observed well when using the comparative phthalocyanine compound. Ta.

[0048]

[Table 2] ──────────────────────────
──────────── Dye for fluorescent labeling

Fluorescence emission (maximum wavelength)──────────────
────────────────────────
Chloroaluminum naphthalocyanine tristle

Sodium phonate
Good detection (~780nm)
Sodium zinc quinocyanine trisulfonate Good detection (~730nm) Chloroaluminum quinoxalocyanine tetra
sodium carboxylate
Good detection (~710nm) Sodium zinc phthalocyanine trisulfonate Undetectable (comparative drug)


Sodium aluminum phthalocyanine disulfonate
Rium (comparative drug)
Undetectable─
──────────────────────────
──────────

[0049]

Effects of the Invention The present invention provides a compact semiconductor laser (67
0 to 780 nm), it was possible to provide an in-vivo diagnostic agent that can easily detect cancer or tumors, and a therapeutic agent for these diseases.

[Brief explanation of the drawing]

[Figure 1] Electronic spectrum of chloroaluminum naphthalocyanine trisulfonic acid sodium salt (~10-6M) in ethanol/water (9/1, weight ratio) (solid line)
and fluorescence emission spectrum (dashed line, excited with 690 nm light)
It is.

[Figure 2] Zinc quinoxalocyanine trisulfonic acid sodium salt (~10-6M) in ethanol/water (9/1
, weight ratio) (solid line) and fluorescence emission spectrum (dashed line, excited with 650 nm light).

[Figure 3] Chloroaluminum quinocyanine trisulfonic acid sodium salt (~10-6M) in ethanol/
Electronic spectrum (solid line) and fluorescence emission spectrum (dashed line, excited with 650 nm light) in water (9/1, weight ratio).

Figure 4: Zinc naphthalocyanine tetracarboxylic acid sodium salt (~10-6M) in ethanol/water (4/1
, weight ratio) (solid line) and fluorescence emission spectrum (dashed line, excited with 690 nm light).

Figure 5: Electronic spectrum (solid line) and fluorescence emission spectrum (dashed line, 650 nm light) of chloraluminum quinoxalocyanine tetracarboxylic acid sodium salt (~10-6M) in ethanol/water (4/1, weight ratio). (excitation).

[Figure 6] [Bis(trimethylsiloxy)silicon naphthalocyanine trisulfonic acid sodium salt (~10-
6M) in ethanol/water (9/1, weight ratio) (solid line) and fluorescence emission spectrum (dashed line, 6M) in ethanol/water (9/1, weight ratio).
(excitation with 90 nm light).

Claims (2)

[Claims] Claim 1: Chemical formula 1 [Chemical formula 1] [In chemical formula 1, M is H2, Al, Si, P, Ga, Ge
, Cd, Sc, Mg, Sn or Zn, R1 , R
2 , R3 and R4 are each independently -XQW
, -QW, -W or a hydrogen atom, X is an oxygen atom,
Nitrogen atom, sulfur atom, phosphorus atom, silicon atom, selenium atom, CR5 R6 (However, R5 and R6 are each independently a hydrogen atom, an alkyl group, an aryl group, or an aralkyl group, and R5 R6 may be carbonyl oxygen. ), or a phenylene group, Q represents a bonding group (linker) between X and W, and W is -OH, -O -
, -SH, -S - , -CO2 H, -CO2 -
, -OCH2 CO2 H, -OCH2 CO2 -
, -PO42 - , -PO3- , -SO3-, -
SO2- , -SO2 Cl, -SO4 2 - , -
NH2 , -NHR7 , -NR8 R9 or -N(
+) R10R11R12 (However, R7 to R12 are
each independently a C1 to C10 alkyl group, a C6 to C10 alkyl group;
It is a C12 aryl group or a C6 to C12 aralkyl group. ), k, l, m and n each independently represent an integer of 0 to 4, Y is a halogen atom, -OR1
3 or -NR142 (However, R13 and R14 are
Each independently a hydrogen atom, an alkyl group that may have a hydrophilic substituent, an acyl group that may have a hydrophilic substituent, and a silyl group that may have a hydrophilic substituent. or a phosphorus atom-containing group which may have a hydrophilic substituent. ), and p is
Indicates an integer from 0 to 2 representing the number of bonds of Y to M, Z1
~Z8 each independently represent a methylene group or a nitrogen atom. ] An in-vivo diagnostic agent for detecting cancer or tumor containing a fluorescent labeling dye. [Claim 2] Chemical formula 2 [Chemical formula 2] [In chemical formula 2, M is H2, Al, Si, P, Ga, Ge
, Cd, Sc, Mg, Sn or Zn, R1 , R
2 , R3 and R4 are each independently -XQW
, -QW, -W or a hydrogen atom, X is an oxygen atom,
Nitrogen atom, sulfur atom, phosphorus atom, silicon atom, selenium atom, CR5 R6 (However, R5 and R6 are each independently a hydrogen atom, an alkyl group, an aryl group, or an aralkyl group, and R5 R6 may be carbonyl oxygen. ), or a phenylene group, Q represents a bonding group (linker) between X and W, and W is -OH, -O -
, -SH, -S - , -CO2 H, -CO2 -
, -OCH2 CO2 H, -OCH2 CO2 -
, -PO42 - , -PO3- , -SO3-, -
SO2- , -SO2 Cl, -SO4 2 - , -
NH2 , -NHR7 , -NR8 R9 or -N(
+) R10R11R12 (However, R7 to R12 are
each independently a C1 to C10 alkyl group, a C6 to C10 alkyl group;
It is a C12 aryl group or a C6 to C12 aralkyl group. ), k, l, m and n each independently represent an integer of 0 to 4, Y is a halogen atom, -OR1
3 or -NR142 (However, R13 and R14 are
Each independently a hydrogen atom, an alkyl group that may have a hydrophilic substituent, an acyl group that may have a hydrophilic substituent, and a silyl group that may have a hydrophilic substituent. or a phosphorus atom-containing group which may have a hydrophilic substituent. ), and p is
Indicates an integer from 0 to 2 representing the number of bonds of Y to M, Z1
~Z8 each independently represent a methylene group or a nitrogen atom. ] A therapeutic drug for cancer or tumor containing a fluorescent labeling dye represented by