Tumor cell killing agent and (tumor cell killing agent)-antibody conjugate

Abstract

This tumor cell killing agent is represented by general formula (I). In general formula (I), X1 to X8 each independently represent a hydrogen atom or a substituent (excluding a halogen atom), wherein X1 and X2, X3 and X4, X5 and X6, and X7 and X8 may be each independently bound to each other via a substituent to form a benzene ring; Y represents an oxygen atom, or a sulfur atom; and R1 to R8 each independently represent an organic group, wherein, when Y is an oxygen atom, then an option where all of R1 to R8 are phenyl groups is excluded.

Description

Tumor cell killing agents and tumor cell killing agent-antibody conjugates

[0001] The present disclosure relates to tumor cell killing agents and tumor cell killing agent-antibody conjugates.

[0002] Singlet oxygen is a type of reactive oxygen and has high reactivity. Utilizing the high reactivity of singlet oxygen, applications to therapeutic drugs, treatment methods, and the development of new drugs are being considered. Phthalocyanine compounds are being considered as one of the materials that generate singlet oxygen (see, for example, Patent Document 1).

[0003] Patent Document 1: Japanese Patent Application Laid-Open No. 2001-064284

[0004] Porphyrinoid compounds, such as phthalocyanine compounds and naphthalocyanine compounds, have an absorption peak in the visible light region, and when porphyrinoid compounds are irradiated with light in this region, singlet oxygen is generated. When porphyrinoid compounds are used as therapeutic agents or treatment methods for diseases, it is preferable that the efficiency of singlet oxygen generation by the porphyrinoid compounds is high. The present disclosure has been made in consideration of the above-mentioned conventional circumstances, and one aspect of the present disclosure aims to provide a tumor cell killing agent and a tumor cell killing agent-antibody conjugate that have excellent singlet oxygen generation efficiency.

[0005] Specific means for achieving the above object are as follows: <1> A tumor cell killing agent represented by the following general formula (I):

[0006]

[0007] (In general formula (I), X １ ~X ８ each independently represents a hydrogen atom or a substituent (excluding a halogen atom). １ and X ２ , X ３ and X ４ , X ５ and X ６ and X ７ and X ８ may each independently be linked to each other via a substituent to form a benzene ring. Y represents an oxygen atom or a sulfur atom. R １ ~R ８each independently represents an organic group, provided that when Y is an oxygen atom, R １ ~R ８ In the general formula (I), X cannot all be phenyl groups. １ ~X ８ <3> The tumor cell killing agent according to <1>, wherein R is a hydrogen atom and Y is an oxygen atom. １ ~R ８ <4> The tumor cell killing agent according to <2>, wherein at least one of the organic groups represented by the general formula (I) has a hydroxy group. １ ~R ８ <5> The tumor cell killing agent according to <2> or <3>, wherein at least one of the organic groups represented by the general formula (I) has a carboxy group or an alkali metal salt thereof. １ ~R ８ <6> The tumor cell killing agent according to any one of <2> to <4>, wherein at least one of the organic groups represented by the general formula (I) has a maleimide group. １ ~R ８ The organic group represented by -(R ９ O) ｎ -R １０ (wherein R ９ represents an alkylene group, and R １０ represents an alkyl group; and n represents an integer of 1 or more. １ ~R ８ <8> The tumor cell killing agent according to <2>, wherein the organic group represented by the formula (I) is an alkyl group having a total of 1 to 18 carbon atoms. １ ~X ８ are each independently an alkoxy group or an aryloxy group, and Y is an oxygen atom. １ ~R ８ <10> The tumor cell killing agent according to <8>, wherein at least one of the organic groups represented by the general formula (I) has a hydroxy group. １ ~R ８<11> The tumor cell killing agent according to <8> or <9>, wherein at least one of the organic groups represented by the general formula (I) has a carboxy group or an alkali metal salt thereof. １ ~R ８ <12> The tumor cell killing agent according to any one of <8> to <10>, wherein at least one of the organic groups represented by the general formula (I) has a maleimide group. １ ~R ８ The organic group represented by -(R ９ O) ｎ -R １０ (wherein R ９ represents an alkylene group, and R １０ represents an alkyl group; and n represents an integer of 1 or more. １ ~X ８ are each independently a thioalkoxy group or a thioaryloxy group, and Y is an oxygen atom. １ and X ２ , X ３ and X ４ , X ５ and X ６ and X ７ and X ８ are linked to each other via a substituent to form a benzene ring, and Y is an oxygen atom. １ ~R ８ <16> The tumor cell killing agent according to <14>, wherein at least one of the organic groups represented by the general formula (I) has a hydroxy group. １ ~R ８ <17> The tumor cell killing agent according to <14> or <15>, wherein at least one of the organic groups represented by the general formula (I) has a carboxy group or an alkali metal salt thereof. １ ~R ８ <18> The tumor cell killing agent according to any one of <14> to <16>, wherein at least one of the organic groups represented by the general formula (I) has a maleimide group. １ ~R８ The organic group represented by -(R ９ O) ｎ -R １０ (wherein R ９ represents an alkylene group, and R １０ represents an alkyl group; and n represents an integer of 1 or more. １ ~X ８ <20> The tumor cell killing agent according to <1>, wherein: is a hydrogen atom, and Y is a sulfur atom. <21> The tumor cell killing agent according to <1>, wherein: ２ The tumor cell killing agent according to any one of <1> to <19>, wherein the carboxyl group or an alkali metal salt thereof of the tumor cell killing agent is O or more. <21> A tumor cell killing agent-antibody conjugate in which the tumor cell killing agent and the antibody are conjugated via an amide bond formed between a carboxyl group or an alkali metal salt thereof carried by the tumor cell killing agent according to <4> and an amino group contained in an antibody having an amino group. <22> A tumor cell killing agent-antibody conjugate in which the tumor cell killing agent and the antibody are conjugated via a thioether bond formed between a maleimide group carried by the tumor cell killing agent according to <5> and a thiol group contained in an antibody having a thiol group. <23> The tumor cell killing agent-antibody conjugate according to <21> or <22>, wherein the antibody is an anti-CD44 rat IgG.

[0008] According to one aspect of the present disclosure, there are provided a tumor cell killing agent and a tumor cell killing agent-antibody conjugate that are highly efficient in generating singlet oxygen.

[0009] This figure shows the evaluation of the cytotoxicity of comparative compound 1, compound 1, and compound 2 without light irradiation. The start of culture is plotted as day 0. This is a representative photograph of tumor cells cultured with the addition of comparative compound 1, compound 1, and compound 2, taken with a 10x objective lens of a phase-contrast microscope. This is a representative photograph of tumor cells cultured with the addition of comparative compound 1, compound 1, and compound 2, taken with a 40x objective lens of a phase-contrast microscope. This is a representative photograph of tumor cells imaged with a phase-contrast microscope one hour after the addition of comparative compound 1, compound 1, and compound 2, followed by irradiation with LED light at a central wavelength of 660 nm. This is a representative photograph of tumor cells imaged with a phase-contrast microscope one hour after the addition of comparative compound 1, compound 1, and compound 2, followed by irradiation with LED light at a central wavelength of 730 nm. This is a representative photograph of tumor cells imaged with a phase-contrast microscope 24 hours after the addition of comparative compound 1, compound 1, and compound 2, followed by irradiation with LED light at a central wavelength of 660 nm. This is a representative photograph of tumor cells photographed with a phase-contrast microscope 24 hours after the addition of comparative compound 1, compound 1, and compound 2, followed by irradiation with LED light at a central wavelength of 730 nm. This is a diagram showing the results of evaluation of cytotoxicity one hour after the addition of comparative compound 1, compound 1, and compound 2, followed by irradiation with LED light at a central wavelength of 660 nm. This is a diagram showing the results of evaluation of cytotoxicity one hour after the addition of comparative compound 1, compound 1, and compound 2, followed by irradiation with LED light at a central wavelength of 730 nm. This is a diagram showing the results of evaluation of cytotoxicity 24 hours after the addition of comparative compound 1, compound 1, and compound 2, followed by irradiation with LED light at a central wavelength of 660 nm. This is a diagram showing the results of evaluation of cytotoxicity 24 hours after the addition of comparative compound 1, compound 1, and compound 2, followed by irradiation with LED light at a central wavelength of 730 nm. This is a representative photograph of tumor cells photographed with a phase-contrast microscope one hour after the addition of a conjugate in which compound 2 is conjugated to an antibody. This is a representative photograph of tumor cells taken with a phase-contrast microscope 24 hours after the addition of a conjugate of Compound 2 to an antibody. This is a representative photograph of tumor cells taken with a phase-contrast microscope 1 hour after the addition of a conjugate of Compound 2 to an antibody and subsequent irradiation with LED light with a central wavelength of 730 nm.Representative photographs of tumor cells photographed with a phase-contrast microscope 24 hours after the addition of a conjugate of compound 2 to an antibody and subsequent irradiation with LED light at a central wavelength of 730 nm. Figure 1 shows the cytotoxicity of tumor cells 24 hours after the addition of a conjugate of compound 2 to an antibody and subsequent irradiation with LED light at a central wavelength of 730 nm, or after non-irradiation. Representative photographs of tumor cells photographed with a phase-contrast microscope 1 hour and 24 hours after the addition of a solution containing compound 2 and various concentrations of DMSO and subsequent irradiation with LED light at a central wavelength of 730 nm. Figure 1 shows the cytotoxicity of tumor cells 24 hours after the addition of a solution containing compound 2 and various concentrations of DMSO and subsequent irradiation with LED light at a central wavelength of 730 nm. Figure 2 shows the cytotoxicity of tumor cells 24 hours after the addition of a solution containing compound 2 and various concentrations of DMSO and subsequent irradiation with LED light at a central wavelength of 730 nm. Figure 3 shows the cytotoxicity of tumor cells 24 hours after the addition of comparative compound 1, compound 1, and compound 2 and subsequent irradiation with LED light at 4, 6, and 12 mW / cm. 2 The tumor cells were irradiated with LED light having a central wavelength of 730 nm at an intensity of 4, 6, and 12 mW / cm 24 hours later, and then photographed with a phase-contrast microscope. 2Figure 1 shows the results of irradiating tumor cells with LED light at a central wavelength of 730 nm at an intensity of 1000 kJ / cm2 and evaluating cytotoxicity after 24 hours. Figure 2 shows representative photographs of tumor cells captured with a phase-contrast microscope after 24 hours of irradiating with LED light at a central wavelength of 730 nm following the addition of 0, 1, 5, and 10 mM NAC, followed by the addition of comparative compound 1, compound 1, and compound 2. Figure 3 shows the results of irradiating tumor cells with LED light at a central wavelength of 730 nm following the addition of 0, 1, 5, and 10 mM NAC, followed by the addition of comparative compound 1, compound 1, and compound 2. Figure 4 shows the results of irradiating tumor cells with LED light at a central wavelength of 730 nm and evaluating cytotoxicity after 24 hours of irradiating tumor cells with LED light at a central wavelength of 730 nm. Figure 5 shows the results of evaluating the labeling efficiency of conjugates at different combinations of pre-reaction time and EDC to compound 2 ratio when the compound 2 to antibody ratio was 200:1 and 20:1. Figure 6 shows the results of evaluating the labeling efficiency of conjugates at different combinations of pre-reaction time and compound 2 to antibody ratio when the EDC to compound 2 ratio was 1:0.5 and 1:1. 1 is a diagram evaluating the absorption spectra of an antibody, comparative compound 1, compound 2, and a conjugate to which comparative compound 1 or compound 2 is conjugated. This is a diagram evaluating the relationship between the compound to antibody ratio and the labeling rate for comparative compound 1 or compound 2. This is a diagram evaluating the breakdown of labeling conditions for comparative compound 1 or compound 2 and the labeling rate of the conjugate under those conditions. This is a diagram evaluating the conjugate to which compound 2, an antibody, and compound 2 are conjugated by SDS-PAGE. This is a diagram evaluating the antigen recognition ability of a conjugate to which an antibody and compound 2 are conjugated by Western blotting. This is a representative photograph obtained by adding a conjugate to which an isotype control, an antibody, and compound 2 are conjugated to tumor cells, followed by fluorescent labeling with a secondary antibody and capturing the image with a confocal microscope. This is a diagram evaluating the antigen recognition ability of a conjugate to which comparative compound 1 or compound 2 is conjugated by Western blotting. A conjugate of Compound 2 conjugated to an antibody was added, followed by irradiation with LED light at a central wavelength of 730 nm. This is a representative photograph of tumor cells taken 24 hours later using a 10x objective lens on a phase-contrast microscope. A conjugate of Compound 2 conjugated to an antibody was added, followed by washing, followed by irradiation with LED light at a central wavelength of 730 nm. This is a representative photograph of tumor cells taken 24 hours later using a 10x objective lens on a phase-contrast microscope.A representative photograph of tumor cells imaged with a 10x objective lens on a phase contrast microscope 24 hours after addition of a conjugate of comparative compound 1 conjugated to an antibody, followed by irradiation with LED light at a central wavelength of 660 nm, was taken. A representative photograph of tumor cells imaged with a 10x objective lens on a phase contrast microscope 24 hours after addition of a conjugate of comparative compound 1 conjugated to an antibody, followed by washing and irradiation with LED light at a central wavelength of 660 nm was taken. A representative photograph of tumor cells imaged with a 40x objective lens on a phase contrast microscope 24 hours after addition of a conjugate of compound 2 conjugated to an antibody, followed by washing and irradiation with LED light at a central wavelength of 730 nm was taken. A representative photograph of tumor cells imaged with a 40x objective lens on a phase contrast microscope 24 hours after addition of a conjugate of compound 2 conjugated to an antibody, followed by washing and irradiation with LED light at a central wavelength of 730 nm was taken.

[0033] Figure 1 shows representative photographs of tumor cells imaged with a 40x objective lens of a phase-contrast microscope 24 hours after addition of a conjugate in which comparative compound 1 is conjugated to an antibody, followed by irradiation with LED light at a central wavelength of 660 nm. Figure 2 shows representative photographs of tumor cells imaged with a 40x objective lens of a phase-contrast microscope 24 hours after addition of a conjugate in which comparative compound 1 is conjugated to an antibody, followed by washing and then irradiation with LED light at a central wavelength of 660 nm. Figure 3 shows the results of evaluation of cytotoxicity 24 hours after addition of a conjugate in which comparative compound 1 or compound 2 is conjugated to an antibody under labeling condition L, followed by irradiation with LED light at a central wavelength of 660 or 730 nm. This shows a sample table for Animal Test 1, and a graph evaluating the labeling rate of the conjugate of Compound 2 conjugated to an antibody under labeling condition H at a scale of 2 nmol or 50 nmol, and the weight change in tumor-inoculated animals administered with PBS, antibody, or the conjugate of Compound 2 conjugated to an antibody under labeling condition H. This shows representative photographs of the liver and kidney of tumor-inoculated animals administered with PBS, antibody, or the conjugate of Compound 2 conjugated to an antibody under labeling condition H, which were excised, stained with H&E, and then photographed with a phase-contrast microscope.This figure shows representative photographs of tumors excised from tumor-inoculated animals administered PBS or antibody, a conjugate of Compound 2 conjugated to an antibody under labeling condition H, and then fluorescently labeled with a secondary antibody, followed by imaging with a 10x objective lens on a confocal microscope. This figure shows representative photographs of tumors excised from tumor-inoculated animals administered PBS or antibody, a conjugate of Compound 2 conjugated to an antibody under labeling condition H, followed by fluorescently labeled with a secondary antibody, followed by imaging with a 20x objective lens on a confocal microscope. This figure shows a sample table for Animal Test 2. This figure shows representative photographs of tumors excised from tumor-inoculated animals administered PBS or antibody, a conjugate of Compound 2 conjugated to an antibody under labeling condition H, followed by imaging with a 20x objective lens on a confocal microscope. This figure shows a sample table for Animal Test 2. This figure shows representative photographs of tumors excised from tumor-inoculated animals during laser irradiation and a table of laser irradiation conditions for Animal Test 2. This figure shows evaluation of tumor weight progression in tumor-inoculated animals listed in the sample table for Animal Test 2, evaluated by fold change, followed by evaluation of tumor weight progression and fold change in tumor-inoculated animals listed in the sample table for Animal Test 2. This figure shows representative photographs of tumors on the laser-irradiated side (right side) of 15 mg / kg αCD44-Compound 2 (2LD, 100 J) 1 hpi and 2LD. Representative photographs of the liver from a 15 mg / kg αCD44-Compound 2 (2LD, 100 J) mouse treated with 15 mg / kg αCD44-Compound 2 (2LD, 100 J) at 1 hpi. Representative H&E stained images of the tumor on the non-laser-irradiated side (left side) from a 15 mg / kg αCD44-Compound 2 (2LD, 100 J) mouse treated with 15 mg / kg αCD44-Compound 2 (2LD, 100 J) at 1 hpi. Representative H&E stained images of the tumor on the laser-irradiated side (right side) from a 15 mg / kg αCD44-Compound 2 (2LD, 100 J) mouse treated with 15 mg / kg αCD44-Compound 2 (2LD, 100 J) at 1 hpi. Fluorescence intensity derived from αCD44-Compound 2 was evaluated in the liver, kidney, and tumor tissues. Representative photographs of the tumor, liver, and kidney from tumor-inoculated animals administered PBS, antibody-conjugates of Compound 2 under labeling condition H, or L were taken with a 10x objective lens on a confocal microscope after fluorescent labeling with secondary antibodies. This is a representative photograph of tumors excised from tumor-injected animals administered a conjugate of compound 2 conjugated to an antibody under labeling condition H, and then fluorescently labeled with a secondary antibody. The photograph was taken with a 10x objective lens on a confocal microscope. This is a graph showing the fluorescence intensity derived from αCD44-compound 2 evaluated in tumor tissue. This is a graph showing the evaluation of the cytotoxicity of compound 4-5 in the absence of light irradiation. The start of culture is plotted as day 0. This is a representative photograph of tumor cells cultured with compound 4-5, taken with a 10x objective lens on a phase-contrast microscope.Representative photographs of tumor cells cultured with the addition of compounds 4-5, taken with a 40x objective lens on a phase-contrast microscope. After the addition of compounds 3-6, cells were irradiated with LED light at a central wavelength of 730 nm, and cytotoxicity was evaluated 1 hour and 24 hours later. After the addition of compounds 3-6, cells were irradiated with LED light at a central wavelength of 730 nm, and cytotoxicity was evaluated relative to the value of DMSO addition 1 hour and 24 hours later. Representative photographs of tumor cells cultured with the addition of compounds 3-6, taken with a 40x objective lens on a phase-contrast microscope. After the addition of compounds 3-6, cells were irradiated with LED light at a central wavelength of 730 nm, and cytotoxicity was evaluated 1 hour and 24 hours later. After the addition of compound 6, cells were irradiated with LED light at a central wavelength of 730 nm, and cytotoxicity was evaluated 1 hour and 24 hours later, and representative photographs of tumor cells cultured with the addition of compound 7 were taken with a 10x objective lens and a 40x objective lens on a phase-contrast microscope. The cytotoxicity of compound 7 was evaluated without light irradiation. The start of culture is plotted as day 0. After the addition of compound 7, tumor cells were irradiated with LED light at a central wavelength of 660 nm. Representative photographs were taken with a phase-contrast microscope of tumor cells 1 hour and 24 hours later. After the addition of compound 7, tumor cells were irradiated with LED light at a central wavelength of 730 nm. Representative photographs were taken with a phase-contrast microscope of tumor cells 1 hour and 24 hours later. After the addition of compound 7, tumor cells were irradiated with LED light at a central wavelength of 660 nm. This figure shows the cytotoxicity evaluation after 1 hour and 24 hours. After the addition of compound 7, tumor cells were irradiated with LED light at a central wavelength of 730 nm. ...

[0010] The present disclosure will be described in detail below. However, the present disclosure is not limited to the following embodiments. In the following embodiments, the components (including element steps, etc.) are not essential unless otherwise specified. The same applies to numerical values ​​and their ranges, and do not limit the present disclosure.

[0011] In the present disclosure, numerical ranges indicated using "to" include the numerical values ​​before and after "to" as the minimum and maximum values, respectively. In numerical ranges described in stages in the present disclosure, the upper or lower limit value described in one numerical range may be replaced with the upper or lower limit value of another staged numerical range. Furthermore, in numerical ranges described in the present disclosure, the upper or lower limit value of that numerical range may be replaced with a value shown in the examples. In the present disclosure, each component may contain multiple corresponding substances. When multiple substances corresponding to each component are present in the composition, the content or amount of each component means the total content or amount of the multiple substances present in the composition, unless otherwise specified. In the present disclosure, the compound represented by the following general formula may be a mixture of positional isomers.

[0012] <Tumor Cell Killing Agent> The tumor cell killing agent of the present disclosure is represented by the following general formula (I).

[0013]

[0014] In general formula (I), X １ ~X ８ each independently represents a hydrogen atom or a substituent (excluding a halogen atom). １ and X ２ , X ３ and X ４ , X ５ and X ６ and X ７ and X ８ may each independently be linked to each other via a substituent to form a benzene ring. Y represents an oxygen atom or a sulfur atom. R １ ~R ８ each independently represents an organic group, provided that when Y is an oxygen atom, R １ ~R ８ Not all of the groups are phenyl groups.

[0015] The tumor cell killing agent represented by general formula (I) is described in detail below. １ ~X ８each independently represents a hydrogen atom or a substituent (excluding a halogen atom). １ ~X ８ The substituent represented by X is not particularly limited. １ ~X ８ Examples of the substituent represented by the formula (I) include an alkyl group, an aryl group, an alkoxy group, an aryloxy group, a thioalkoxy group, a thioaryloxy group, a hydroxy group, a mercapto group, an amino group, a ureido group, a formyl group, an acyl group, a carboxy group or an alkali metal salt thereof, a cyano group, a nitro group, a sulfo group or an alkali metal salt thereof, -(R ９ O) ｎ -R １０ (wherein R ９ represents an alkylene group, and R １０ represents an alkyl group. n represents an integer of 1 or more. Hereinafter, this may be referred to as a specific alkylated alkyleneoxy group.) or a substituent formed by combining two or more of these substituents. ９ The alkylene group represented by the formula (I) is preferably an alkylene group having 2 to 6 carbon atoms, and examples thereof include an ethylene group, a propylene group, a trimethylene group, a tetramethylene group, a pentamethylene group, and a hexamethylene group. １０ The alkyl group represented by the formula (I) is preferably an alkyl group having 1 to 6 carbon atoms, and examples thereof include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a t-butyl group, an n-pentyl group, an isopentyl group, a sec-pentyl group, a t-pentyl group, and an n-hexyl group. １ ~X ８ is preferably a hydrogen atom, an alkoxy group, an aryloxy group, a thioalkoxy group, or a thioaryloxy group. These substituents may further have a substituent. １ ~X ８Substituents that further substitute the substituent represented by the formula (I) may include a hydroxy group; an amino group; a carboxy group; a mercapto group; a ureido group; a group having a hydroxy group; a group having a carboxy group or an alkali metal salt thereof; a group having a maleimide group; an alkoxy group; a halogen atom, etc. The hydroxy group, amino group, carboxy group, etc. may be protected by a protecting group. Examples of the protecting group for the hydroxy group include a tert-butyldimethylsilyl (TBDMS) group, etc.

[0016] In general formula (I), X １ and X ２ , X ３ and X ４ , X ５ and X ６ and X ７ and X ８ may each independently be linked to each other via a substituent to form a benzene ring. １ and X ２ , X ３ and X ４ , X ５ and X ６ and X ７ and X ８ It is preferred that all of the groups be linked to each other via a substituent to form a benzene ring.

[0017] In general formula (I), R １ ~R ８ each independently represents an organic group, provided that when Y is an oxygen atom, R １ ~R ８ Not all of R are phenyl groups. １ ~R ８ The organic group represented by R is not particularly limited. １ ~R ８ The organic group represented by the formula (I) is the above-mentioned X １ ~X ８ Among the specific examples of the substituent represented by the formula (I) or a substituent formed by combining two or more of the above-mentioned substituents, a group in which the position bonded to Y is a carbon atom can be mentioned.

[0018] In the tumor cell killing agent represented by general formula (I), X １ ~X ８ , Y and R１ ~R ８ The combination of X in the general formula (I) is not particularly limited. １ ~X ８ may be a combination in which X is a hydrogen atom and Y is an oxygen atom; １ ~X ８ may be a combination in which each independently represents an alkoxy group or an aryloxy group, and Y represents an oxygen atom; １ ~X ８ may be a combination in which each independently represents a thioalkoxy group or a thioaryloxy group, and Y represents an oxygen atom; １ and X ２ , X ３ and X ４ , X ５ and X ６ and X ７ and X ８ may be a combination in which X are linked to each other via a substituent to form a benzene ring, and Y is an oxygen atom; １ ~X ８ may be a combination in which X is a hydrogen atom and Y is a sulfur atom; １ ~X ８ may be a combination in which each independently represents an alkoxy group or an aryloxy group, and Y represents a sulfur atom, １ ~X ８ may be a combination in which each independently represents a thioalkoxy group or a thioaryloxy group, and Y represents a sulfur atom, １ and X ２ , X ３ and X ４ , X ５ and X ６ and X ７ and X ８ may be a combination in which each of them is linked to each other via a substituent to form a benzene ring, and Y is a sulfur atom. １ ~X ８ and Y, R １ ~R ８ is not particularly limited.

[0019] (First embodiment) The tumor cell killing agent of the first embodiment represented by general formula (I) is １ ~X ８ is a hydrogen atom, Y is an oxygen atom, and R １ ~R ８ At least one of the organic groups represented by R has a hydroxy group. １ ~R ８ Examples of the organic group having a hydroxy group represented by the formula (I) include a group in which a hydrogen atom of an alkyl group is substituted with a hydroxy group. In this case, the alkyl group is preferably an alkyl group having a total of 1 to 4 carbon atoms, more preferably an ethyl group. In the tumor cell killing agent of the first embodiment, R １ ~R ８ At least one of the organic groups represented by R １ ~R ８ All of the organic groups represented by R may be groups having a hydroxy group. １ ~R ８ When the organic group represented by R １ ~R ８ Examples of the organic group represented by the formula (I) include an alkyl group, an aryl group, and a specific alkylated alkyleneoxy group, with the specific alkylated alkyleneoxy group and an alkyl group being preferred, and the specific alkylated alkyleneoxy group and an alkyl group having a total carbon number of 1 to 4 being more preferred. Specific examples of the tumor cell killing agent of the first embodiment are shown below, but the tumor cell killing agent of the first embodiment is not limited to the specific examples below.

[0020]

[0021] The tumor cell killing agent of the first embodiment is R １ ~R ８At least a portion of the hydroxy groups contained in the organic group represented by the formula (I) may be protected with a protecting group. Specific examples of the tumor cell killing agent of the first embodiment in which the hydroxy groups are protected with a protecting group are shown below, but the tumor cell killing agent of the first embodiment in which the hydroxy groups are protected with a protecting group is not limited to the following specific examples. In the following specific examples, TBDMS represents a tert-butyldimethylsilyl group.

[0022]

[0023] (Second embodiment) In the second embodiment of the tumor cell killing agent represented by general formula (I), X １ ~X ８ is a hydrogen atom, Y is an oxygen atom, and R １ ~R ８ At least one of the organic groups represented by R is a group having a carboxy group or an alkali metal salt thereof. １ ~R ８ Examples of the organic group having a carboxy group or an alkali metal salt thereof represented by the formula (I) include a group obtained by reacting a group in which an alkyl group is substituted with a hydroxy group with a carboxylic acid anhydride, or an alkali metal salt thereof. In this case, the alkyl group is preferably an alkyl group having a total of 1 to 4 carbon atoms, more preferably an ethyl group. Examples of the carboxylic acid anhydride include succinic anhydride, maleic anhydride, and phthalic anhydride. In the tumor cell killing agent of the second embodiment, R １ ~R ８ At least one of the organic groups represented by R １ ~R ８ All of the organic groups represented by R may be groups having a carboxy group or an alkali metal salt thereof. １ ~R ８ When the organic group represented by the formula (I) has a group other than a carboxy group or a group having an alkali metal salt thereof, R １ ~R ８Examples of the organic group represented by the formula (I) include an alkyl group, an aryl group, and a specific alkylated alkyleneoxy group, with a specific alkylated alkyleneoxy group and an alkyl group being preferred, and a specific alkylated alkyleneoxy group and an alkyl group having a total of 1 to 4 carbon atoms being more preferred. Specific examples of the tumor cell killing agent of the second embodiment are shown below, but the tumor cell killing agent of the second embodiment is not limited to the specific examples below. In addition, the tumor cell killing agent of the second embodiment may form a tumor cell killing agent-antibody complex, as described below, with an antibody via a carboxy group or an alkali metal salt thereof contained in the tumor cell killing agent of the second embodiment.

[0024]

[0025] (Third embodiment) In the tumor cell killing agent of the third embodiment represented by general formula (I), in general formula (I), X １ ~X ８ is a hydrogen atom, Y is an oxygen atom, and R １ ~R ８ At least one of the organic groups represented by R １ ~R ８ Examples of the organic group having a maleimide group represented by the formula (I) include a group in which a structure having a maleimide group is bonded via an ester to a group in which a hydrogen atom of an alkyl group is substituted with a hydroxy group. For example, an organic group having a maleimide group is formed by reacting a group in which a hydrogen atom of an alkyl group is substituted with a hydroxy group with 3-maleimidopropionic acid or the like. In the tumor cell killing agent of the third embodiment, R １ ~R ８ At least one of the organic groups represented by R １ ~R ８ All of the organic groups represented by R may be groups having a maleimide group. １ ~R ８ is a group other than a group having a maleimide group, R １ ~R ８Examples of the organic group represented by the formula (I) include an alkyl group, an aryl group, and a specific alkylated alkyleneoxy group, with the specific alkylated alkyleneoxy group and an alkyl group being preferred, and the specific alkylated alkyleneoxy group and an alkyl group having a total of 1 to 4 carbon atoms being more preferred. Specific examples of the tumor cell killing agent of the third embodiment are shown below, but the tumor cell killing agent of the third embodiment is not limited to the specific examples below. Furthermore, the tumor cell killing agent of the third embodiment may form a tumor cell killing agent-antibody complex, as described below, with an antibody via a maleimide group contained in the tumor cell killing agent of the third embodiment.

[0026]

[0027] (Fourth embodiment) In the fourth embodiment of the tumor cell killing agent represented by general formula (I), １ ~X ８ is a hydrogen atom, Y is an oxygen atom, and R １ ~R ８ The organic group represented by the formula (I) is a combination of specific alkylated alkyleneoxy groups. １ ~R ８ The specific alkylated alkyleneoxy groups represented by the formula (I) may all be the same or different. ９ As R in the specific alkylated alkyleneoxy group, an ethylene group or a trimethylene group is preferable. １０ is preferably a methyl group or an ethyl group. n in the specific alkylated alkyleneoxy group is preferably an integer of 1 to 6, more preferably an integer of 1 to 5, and even more preferably an integer of 1 to 4. Specific examples of the tumor cell killing agent of the fourth embodiment are shown below, but the tumor cell killing agent of the fourth embodiment is not limited to the specific examples below.

[0028]

[0029]

[0030]

[0031] (Fifth embodiment) In the fifth embodiment of the tumor cell killing agent represented by general formula (I), １ ~X ８ is a hydrogen atom, Y is an oxygen atom, and R １ ~R ８ The organic group represented by the formula (I) is a combination of alkyl groups having a total of 1 to 18 carbon atoms. １ ~R ８ Examples of the alkyl group having a total of 1 to 18 carbon atoms represented by the formula (R) include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a t-butyl group, an n-pentyl group, an isopentyl group, a sec-pentyl group, a t-pentyl group, an n-hexyl group, an n-heptyl group, an isoheptyl group, a sec-heptyl group, a 2,4-dimethylpentyl group, an n-octyl group, a 2-ethylhexyl group, and a cyclohexyl group. １ ~R ８ The alkyl group having a total of 1 to 18 carbon atoms, represented by the formula (I), preferably has 2 to 10 carbon atoms in total, and more preferably has 4 to 8 carbon atoms in total.

[0032] (Sixth embodiment) In the sixth embodiment of the tumor cell killing agent represented by general formula (I), １ ~X ８ are each independently an alkoxy group or an aryloxy group, and Y is an oxygen atom. １ ~R ８ The organic group represented by X is not particularly limited. １ ~X ８ Examples of the alkoxy group represented by X include a methoxy group, an ethoxy group, an n-propoxy group, an i-propoxy group, an n-butoxy group, an n-amyloxy group, an n-octyloxy group, and an n-decyloxy group. １ ~X ８ The alkoxy group represented by the following formula may further have a substituent. １ ~X ８Substituents that further substitute the alkoxy group represented by the formula (I) may include a hydroxy group, an amino group, a carboxy group, a mercapto group, a ureido group, a group having a hydroxy group, a group having a carboxy group or an alkali metal salt thereof, a group having a maleimide group, an alkoxy group, a halogen atom, etc. The hydroxy group, amino group, carboxy group, etc. may be protected by a protecting group. An example of a protecting group for a hydroxy group is a tert-butyldimethylsilyl (TBDMS) group, etc. X １ ~X ８ Examples of the aryloxy group represented by X include a phenoxy group and a naphthoxy group. １ ~X ８ The aryloxy group represented by the following formula may further have a substituent. １ ~X ８ Substituents that further substitute the aryloxy group represented by the formula (I) may include a hydroxy group, an amino group, a carboxy group, a mercapto group, a ureido group, a group having a hydroxy group, a group having a carboxy group or an alkali metal salt thereof, a group having a maleimide group, an alkoxy group, a halogen atom, etc. The hydroxy group, amino group, carboxy group, etc. may be protected by a protecting group. An example of a protecting group for a hydroxy group is a tert-butyldimethylsilyl (TBDMS) group, etc. X １ ~X ８ The substituent represented by R is preferably an alkoxy group having a substituent. １ ~R ８ The organic group represented by the formula (I) is preferably a specific alkylated alkyleneoxy group, a group having a hydroxy group, a group having a carboxy group or an alkali metal salt thereof, or a group having a maleimide group, and more preferably a specific alkylated alkyleneoxy group.

[0033] In the tumor cell killing agent of the sixth embodiment represented by general formula (I), R １ ~R ８ At least one of the organic groups represented by the general formula (I) may be a group having a hydroxy group. １ ~R ８The organic group having a hydroxy group represented by the formula (I) includes a group in which a hydrogen atom of an alkyl group is substituted with a hydroxy group. In this case, the alkyl group is preferably an alkyl group having a total of 1 to 4 carbon atoms, more preferably an ethyl group. In the tumor cell killing agent of the sixth embodiment, R １ ~R ８ At least one of the organic groups represented by R may be a group having a hydroxy group, １ ~R ８ All of the organic groups represented by R may be groups having a hydroxy group. １ ~R ８ When the organic group represented by R １ ~R ８ Examples of the organic group represented by R include an alkyl group, an aryl group, and a specific alkylated alkyleneoxy group, with the specific alkylated alkyleneoxy group and an alkyl group being preferred, and the specific alkylated alkyleneoxy group and an alkyl group having a total carbon number of 1 to 4 being more preferred. １ ~R ８ At least a part of the hydroxy groups contained in the organic group represented by the formula (I) may be protected with a protecting group. Examples of the protecting group for the hydroxy group include a tert-butyldimethylsilyl (TBDMS) group.

[0034] In the tumor cell killing agent of the sixth embodiment represented by general formula (I), R １ ~R ８ At least one of the organic groups represented by the general formula (I) may be a group having a carboxy group or an alkali metal salt thereof. １ ~R ８ Examples of the group having a carboxy group represented by the formula (I) or an alkali metal salt thereof include a group obtained by reacting a carboxylic acid anhydride with a group in which an alkyl group is substituted with a hydroxy group, or an alkali metal salt thereof. In this case, the alkyl group is preferably an alkyl group having a total of 1 to 4 carbon atoms, more preferably an ethyl group. Examples of the carboxylic acid anhydride include succinic anhydride, maleic anhydride, and phthalic anhydride. In the tumor cell killing agent of the sixth embodiment, R１ ~R ８ At least one of the organic groups represented by R may be a group having a carboxy group or an alkali metal salt thereof, １ ~R ８ All of the organic groups represented by R may be groups having a carboxy group or an alkali metal salt thereof. １ ~R ８ When the organic group represented by the formula (I) has a group other than a carboxy group or a group having an alkali metal salt thereof, R １ ~R ８ Examples of the organic group represented by the formula (I) include an alkyl group, an aryl group, and a specific alkylated alkyleneoxy group. Of these, the specific alkylated alkyleneoxy group and an alkyl group are preferred, and the specific alkylated alkyleneoxy group and an alkyl group having a total of 1 to 4 carbon atoms are more preferred.

[0035] In the tumor cell killing agent of the sixth embodiment represented by general formula (I), R １ ~R ８ At least one of the organic groups represented by the general formula (I) may be a group having a maleimide group. １ ~R ８ Examples of the organic group having a maleimide group represented by the formula (I) include a group in which a structure having a maleimide group is bonded via an ester to a group in which a hydrogen atom of an alkyl group is substituted with a hydroxy group. １ ~R ８ At least one of the organic groups represented by R may be a group having a maleimide group, １ ~R ８ All of the organic groups represented by R may be groups having a maleimide group. １ ~R ８ is a group other than a group having a maleimide group, R １ ~R ８ Examples of the organic group represented by the formula (I) include an alkyl group, an aryl group, and a specific alkylated alkyleneoxy group. Of these, the specific alkylated alkyleneoxy group and an alkyl group are preferred, and the specific alkylated alkyleneoxy group and an alkyl group having a total of 1 to 4 carbon atoms are more preferred.

[0036] In the tumor cell killing agent of the sixth embodiment represented by general formula (I), R １ ~R ８ The organic group represented by the formula (I) may be a specific alkylated alkyleneoxy group. ９ As R in the specific alkylated alkyleneoxy group, an ethylene group or a trimethylene group is preferable. １０ As for n in the specific alkylated alkyleneoxy group, an integer of 1 to 6 is preferred, an integer of 1 to 4 is more preferred, and an integer of 1 to 2 is even more preferred. １ ~R ８ The specific alkylated alkyleneoxy groups represented by the formula (I) may all be the same or different.

[0037] Specific examples of the tumor cell killing agent of the sixth embodiment are shown below, but the tumor cell killing agent of the sixth embodiment is not limited to the following specific examples. In the following specific examples, Ph represents a phenyl group.

[0038]

[0039]

[0040]

[0041] (Seventh embodiment) In the seventh embodiment of the tumor cell killing agent represented by general formula (I), １ ~X ８ In the seventh embodiment, R is a thioalkoxy group or a thioaryloxy group, and Y is an oxygen atom. １ ~R ８ The organic group represented by X is not particularly limited. １ ~X ８ Examples of the thioalkoxy group represented by the formula (X) include a thiomethoxy group, a thioethoxy group, a thio-n-propoxy group, a thioisopropoxy group, a thio-n-butoxy group, and a thio-t-butoxy group. １ ~X ８The thioalkoxy group represented by the following formula may further have a substituent. １ ~X ８ Substituents that further substitute the thioalkoxy group represented by the formula (I) may include a hydroxy group, an amino group, a carboxy group, a mercapto group, a ureido group, a group having a hydroxy group, a group having a carboxy group or an alkali metal salt thereof, a group having a maleimide group, an alkoxy group, a halogen atom, etc. The hydroxy group, amino group, carboxy group, etc. may be protected by a protecting group. An example of a protecting group for a hydroxy group is a tert-butyldimethylsilyl (TBDMS) group, etc. X １ ~X ８ Examples of the thioaryloxy group represented by the formula (X) include a thiophenoxy group and a thionaphthoxy group. １ ~X ８ The thioaryloxy group represented by the following formula may further have a substituent. １ ~X ８ Substituents that further substitute the thioaryloxy group represented by the formula (I) may include a hydroxy group, an amino group, a carboxy group, a mercapto group, a ureido group, a group having a hydroxy group, a group having a carboxy group or an alkali metal salt thereof, a group having a maleimide group, an alkoxy group, a halogen atom, etc. The hydroxy group, amino group, carboxy group, etc. may be protected by a protecting group. An example of a protecting group for a hydroxy group is a tert-butyldimethylsilyl (TBDMS) group, etc. X １ ~X ８ The substituent represented by R is preferably a thioaryloxy group, more preferably a thiophenoxy group. １ ~R ８ The organic group represented by the formula (I) is preferably a specific alkylated alkyleneoxy group, a group having a hydroxy group, a group having a carboxy group or an alkali metal salt thereof, or a group having a maleimide group, and more preferably a specific alkylated alkyleneoxy group.

[0042] In the seventh embodiment of the tumor cell killing agent represented by general formula (I), R １ ~R ８In the seventh embodiment of the tumor cell killing agent represented by general formula (I), at least one of the organic groups represented by R １ ~R ８ The organic group having a hydroxy group represented by the formula (I) includes a group in which a hydrogen atom of an alkyl group is substituted with a hydroxy group. In this case, the alkyl group is preferably an alkyl group having a total of 1 to 4 carbon atoms, more preferably an ethyl group. In the tumor cell killing agent of the seventh embodiment, R １ ~R ８ At least one of the organic groups represented by R may be a group having a hydroxy group, １ ~R ８ All of the organic groups represented by R may be groups having a hydroxy group. １ ~R ８ When the organic group represented by R １ ~R ８ Examples of the organic group represented by the formula (I) include an alkyl group, an aryl group, and a specific alkylated alkyleneoxy group, with the specific alkylated alkyleneoxy group and an alkyl group being preferred, and the specific alkylated alkyleneoxy group and an alkyl group having a total carbon number of 1 to 4 being more preferred. １ ~R ８ At least a part of the hydroxy groups contained in the organic group represented by the formula (I) may be protected with a protecting group. Examples of the protecting group for the hydroxy group include a tert-butyldimethylsilyl (TBDMS) group.

[0043] In the seventh embodiment of the tumor cell killing agent represented by general formula (I), R １ ~R ８ In the seventh embodiment of the tumor cell killing agent represented by general formula (I), at least one of the organic groups represented by R １ ~R ８Examples of the group having a carboxy group represented by the formula (I) or an alkali metal salt thereof include a group obtained by reacting a carboxylic acid anhydride with a group in which an alkyl group is substituted with a hydroxy group, or an alkali metal salt thereof. In this case, the alkyl group is preferably an alkyl group having a total of 1 to 4 carbon atoms, more preferably an ethyl group. Examples of the carboxylic acid anhydride include succinic anhydride, maleic anhydride, and phthalic anhydride. In the tumor cell killing agent of the seventh embodiment, R １ ~R ８ At least one of the organic groups represented by R may be a group having a carboxy group or an alkali metal salt thereof, １ ~R ８ All of the organic groups represented by R may be groups having a carboxy group or an alkali metal salt thereof. １ ~R ８ When the organic group represented by the formula (I) has a group other than a carboxy group or a group having an alkali metal salt thereof, R １ ~R ８ Examples of the organic group represented by the formula (I) include an alkyl group, an aryl group, and a specific alkylated alkyleneoxy group. Of these, the specific alkylated alkyleneoxy group and an alkyl group are preferred, and the specific alkylated alkyleneoxy group and an alkyl group having a total of 1 to 4 carbon atoms are more preferred.

[0044] In the seventh embodiment of the tumor cell killing agent represented by general formula (I), R １ ~R ８ At least one of the organic groups represented by the general formula (I) may be a group having a maleimide group. １ ~R ８ Examples of the organic group having a maleimide group represented by the formula (I) include a group in which a structure having a maleimide group is bonded via an ester to a group in which a hydrogen atom of an alkyl group is substituted with a hydroxy group. １ ~R ８ At least one of the organic groups represented by R may be a group having a maleimide group, １ ~R ８ All of the organic groups represented by R may be groups having a maleimide group.１ ~R ８ is a group other than a group having a maleimide group, R １ ~R ８ Examples of the organic group represented by the formula (I) include an alkyl group, an aryl group, and a specific alkylated alkyleneoxy group. Of these, the specific alkylated alkyleneoxy group and an alkyl group are preferred, and the specific alkylated alkyleneoxy group and an alkyl group having a total of 1 to 4 carbon atoms are more preferred.

[0045] In the seventh embodiment of the tumor cell killing agent represented by general formula (I), R １ ~R ８ The organic group represented by the formula (I) may be a specific alkylated alkyleneoxy group. ９ As R in the specific alkylated alkyleneoxy group, an ethylene group or a trimethylene group is preferable. １０ As for n in the specific alkylated alkyleneoxy group, an integer of 1 to 6 is preferred, an integer of 1 to 4 is more preferred, and an integer of 1 to 2 is even more preferred. １ ~R ８ The specific alkylated alkyleneoxy groups represented by the formula (I) may all be the same or different.

[0046] Specific examples of the tumor cell killing agent of the seventh embodiment are shown below, but the tumor cell killing agent of the seventh embodiment is not limited to the following specific examples. In the following specific examples, Ph represents a phenyl group.

[0047]

[0048]

[0049]

[0050] (Eighth embodiment) In the eighth embodiment of the tumor cell killing agent represented by general formula (I), １ and X ２ , X ３ and X ４ , X ５ and X ６ and X ７ and X８ are linked to each other via a substituent to form a benzene ring, and Y is an oxygen atom. １ ~R ８ The organic group represented by X is not particularly limited. １ and X ２ , X ３ and X ４ , X ５ and X ６ and X ７ and X ８ When each of R and R forms a benzene ring, the tumor cell killing agent of the eighth embodiment corresponds to a naphthalocyanine compound. The hydrogen atoms on the formed benzene ring may be substituted with a hydroxy group, an amino group, a carboxy group, a mercapto group, a ureido group, a group having a hydroxy group, a group having a carboxy group or an alkali metal salt thereof, a group having a maleimide group, an alkoxy group, a halogen atom, or the like. The hydroxy group, amino group, carboxy group, or the like may be protected with a protecting group. An example of a protecting group for a hydroxy group is a tert-butyldimethylsilyl (TBDMS) group. R １ ~R ８ Examples of the organic group represented by the formula (I) include a group having a hydroxy group, a group having a carboxy group or an alkali metal salt thereof, a group having a maleimide group, an alkyl group, an aryl group, and a specific alkylated alkyleneoxy group.

[0051] In the eighth embodiment of the tumor cell killing agent represented by general formula (I), R １ ~R ８ At least one of the organic groups represented by the general formula (I) may be a group having a hydroxy group. １ ~R ８ The organic group having a hydroxy group represented by the formula (I) includes a group in which a hydrogen atom of an alkyl group is substituted with a hydroxy group. In this case, the alkyl group is preferably an alkyl group having a total of 1 to 4 carbon atoms, more preferably an ethyl group. In the tumor cell killing agent of the eighth embodiment, R １ ~R ８At least one of the organic groups represented by R may be a group having a hydroxy group, １ ~R ８ All of the organic groups represented by R may be groups having a hydroxy group. １ ~R ８ When the organic group represented by R １ ~R ８ Examples of the organic group represented by the formula (I) include an alkyl group, an aryl group, and a specific alkylated alkyleneoxy group, with the specific alkylated alkyleneoxy group and an alkyl group being preferred, and the specific alkylated alkyleneoxy group and an alkyl group having a total carbon number of 1 to 4 being more preferred. １ ~R ８ At least a part of the hydroxy groups contained in the organic group represented by the formula (I) may be protected with a protecting group. Examples of the protecting group for the hydroxy group include a tert-butyldimethylsilyl (TBDMS) group.

[0052] In the eighth embodiment of the tumor cell killing agent represented by general formula (I), R １ ~R ８ In the eighth embodiment of the tumor cell killing agent represented by general formula (I), at least one of the organic groups represented by R １ ~R ８ Examples of the group having a carboxy group represented by the formula (I) or an alkali metal salt thereof include a group obtained by reacting a carboxylic acid anhydride with a group in which an alkyl group is substituted with a hydroxy group, or an alkali metal salt thereof. In this case, the alkyl group is preferably an alkyl group having a total of 1 to 4 carbon atoms, more preferably an ethyl group. Examples of the carboxylic acid anhydride include succinic anhydride, maleic anhydride, and phthalic anhydride. In the tumor cell killing agent of the eighth embodiment, R １ ~R ８ At least one of the organic groups represented by R may be a group having a carboxy group or an alkali metal salt thereof, １ ~R ８ All of the organic groups represented by R may be groups having a carboxy group or an alkali metal salt thereof.１ ~R ８ When the organic group represented by the formula (I) has a group other than a carboxy group or a group having an alkali metal salt thereof, R １ ~R ８ Examples of the organic group represented by the formula (I) include an alkyl group, an aryl group, and a specific alkylated alkyleneoxy group. Of these, the specific alkylated alkyleneoxy group and an alkyl group are preferred, and the specific alkylated alkyleneoxy group and an alkyl group having a total of 1 to 4 carbon atoms are more preferred.

[0053] In the eighth embodiment of the tumor cell killing agent represented by general formula (I), R １ ~R ８ At least one of the organic groups represented by the general formula (I) may be a group having a maleimide group. １ ~R ８ Examples of the organic group having a maleimide group represented by the formula (I) include a group in which a structure having a maleimide group is bonded via an ester to a group in which a hydrogen atom of an alkyl group is substituted with a hydroxy group. １ ~R ８ At least one of the organic groups represented by R may be a group having a maleimide group, １ ~R ８ All of the organic groups represented by R may be groups having a maleimide group. １ ~R ８ is a group other than a group having a maleimide group, R １ ~R ８ Examples of the organic group represented by the formula (I) include an alkyl group, an aryl group, and a specific alkylated alkyleneoxy group. Of these, the specific alkylated alkyleneoxy group and an alkyl group are preferred, and the specific alkylated alkyleneoxy group and an alkyl group having a total of 1 to 4 carbon atoms are more preferred.

[0054] In the eighth embodiment of the tumor cell killing agent represented by general formula (I), R １ ~R ８ In the eighth embodiment, at least one of the organic groups represented by R１ ~R ８ Preferably, at least one of the organic groups represented by R is a specific alkylated alkyleneoxy group. １ ~R ８ All of the organic groups represented by the formula (I) are specific alkylated alkyleneoxy groups (i.e., in the general formula (I), R １ ~R ８ It is more preferable that the organic group represented by the formula (I) is a specific alkylated alkyleneoxy group. ９ As R in the specific alkylated alkyleneoxy group, an ethylene group or a trimethylene group is preferable. １０ As for n in the specific alkylated alkyleneoxy group, an integer of 1 to 6 is preferred, an integer of 1 to 5 is more preferred, and an integer of 1 to 4 is even more preferred. １ ~R ８ When the organic group represented by R １ ~R ８ Examples of the organic group represented by R include an alkyl group, an aryl group, and a group having a maleimide group. １ ~R ８ The specific alkylated alkyleneoxy groups represented by the following formula (I) may all be the same or different. Specific examples of the tumor cell killing agent of the eighth embodiment are shown below, but the tumor cell killing agent of the eighth embodiment is not limited to the following specific examples.

[0055]

[0056]

[0057]

[0058]

[0059] (Ninth embodiment) In the ninth embodiment of the tumor cell killing agent represented by general formula (I), １ ~X ８ In the ninth embodiment, the tumor cell killing agent is a combination in which R is a hydrogen atom and Y is a sulfur atom. １~R ８ The organic group represented by R is not particularly limited. １ ~R ８ Examples of the organic group represented by R include a group having a hydroxy group, a group having a carboxy group or an alkali metal salt thereof, a group having a maleimide group, an alkyl group, an aryl group, and a specific alkylated alkyleneoxy group. Among these, an alkyl group or an aryl group is preferred, and an aryl group is more preferred. １ ~R ８ Examples of the alkyl group represented by the formula (I) include alkyl groups having a total of 1 to 18 carbon atoms, and specific examples thereof include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a t-butyl group, an n-pentyl group, an isopentyl group, a sec-pentyl group, a t-pentyl group, an n-hexyl group, an n-heptyl group, an isoheptyl group, a sec-heptyl group, a 2,4-dimethylpentyl group, an n-octyl group, a 2-ethylhexyl group, and a cyclohexyl group. １ ~R ８ Examples of the aryl group represented by R include a phenyl group and a naphthyl group. １ ~R ８ The organic group represented by the formula (I) is preferably an aryl group, more preferably a phenyl group. Specific examples of the tumor cell killing agent of the ninth embodiment are shown below, but the tumor cell killing agent of the ninth embodiment is not limited to the following specific examples. In the following specific examples, Ph represents a phenyl group.

[0060]

[0061] (Tenth embodiment) In the tenth embodiment of the tumor cell killing agent represented by general formula (I), １ ~X ８ are each independently an alkoxy group or an aryloxy group, and Y is a sulfur atom. １ ~R ８ The organic group represented by X in the compound of the tenth embodiment is not particularly limited. １ ~X ８ and an alkoxy group or aryloxy group represented by R １ ~R８ The details, preferred ranges, preferred combinations, etc. of the organic group represented by the formula (I) are the same as those in the sixth embodiment. Specific examples of the tumor cell killing agent of the tenth embodiment include compounds in which the oxygen atom corresponding to Y in general formula (I) of the specific examples of the tumor cell killing agent of the sixth embodiment has been substituted with a sulfur atom.

[0062] (Eleventh embodiment) In the eleventh embodiment of the tumor cell killing agent represented by general formula (I), １ ~X ８ In an eleventh embodiment, R is a combination in which each independently represents a thioalkoxy group or a thioaryloxy group, and Y is a sulfur atom. １ ~R ８ The organic group represented by X in the compound of the eleventh embodiment is not particularly limited. １ ~X ８ and a thioalkoxy group or a thioaryloxy group represented by R １ ~R ８ The details, preferred ranges, preferred combinations, etc. of the organic group represented by the formula (I) are the same as those in the seventh embodiment. Specific examples of the tumor cell killing agent of the eleventh embodiment include compounds in which the oxygen atom corresponding to Y in general formula (I) in the specific examples of the tumor cell killing agent of the seventh embodiment has been substituted with a sulfur atom.

[0063] (Twelfth embodiment) In the twelfth embodiment of the tumor cell killing agent represented by general formula (I), １ and X ２ , X ３ and X ４ , X ５ and X ６ and X ７ and X ８ are linked to each other via a substituent to form a benzene ring, and Y is a sulfur atom. １ ~R ８ The organic group represented by X in the compound of the twelfth embodiment is not particularly limited. １ ~X ８ and a benzene ring represented by R １ ~R８ The details, preferred ranges, preferred combinations, etc. of the organic group represented by are the same as those in the above-mentioned eighth embodiment. Specific examples of the tumor cell killing agent of the twelfth embodiment include compounds in which the oxygen atom corresponding to Y in general formula (I) in the specific examples of the tumor cell killing agent of the eighth embodiment has been substituted with a sulfur atom.

[0064] (Solubility) The tumor cell killing agent represented by general formula (I) has a solubility in water at 25°C of 0.1 g / 100 g-H in order to enable its use as an aqueous solution. ２ It is preferable that the density is 0 or more, and 0.3 g / 100 g-H ２ More preferably, it is 0 or more, and 0.5 g / 100 g-H ２ The solubility of the tumor cell killing agent represented by general formula (I) in water at 25°C is preferably 0.1 g / 100 g-H. ２ From the viewpoint of being O or more, in the general formula (I), X １ ~X ８ or a substituent represented by R １ ~R ８ It is preferable that the organic group represented by the formula (I) has a carboxy group or an alkali metal salt thereof, or contains a specific alkylated alkyleneoxy group. In the present disclosure, the solubility in water at 25°C refers to the concentration at which the dissolution of the compound in water is confirmed by visual observation.

[0065] The tumor cell killing agent represented by general formula (I) can be produced by a standard method. The tumor cell killing agent represented by general formula (I) can be synthesized by referring to the methods described in, for example, JP-A-3-62878, JP-A-3-215466, and JP-A-4-226390.

[0066] The tumor cell killing agent represented by general formula (I) exhibits a high quantum yield of singlet oxygen production and is applicable to photodynamic therapy (PDT), photoimmunotherapy (PIT), and the like.

[0067] <Tumor cell killing agent-antibody conjugate> The first tumor cell killing agent-antibody conjugate of the present disclosure is a tumor cell killing agent conjugated to an antibody via an amide bond formed between a carboxy group or an alkali metal salt thereof possessed by the tumor cell killing agent of the second embodiment and an amino group contained in an antibody having an amino group. The second tumor cell killing agent-antibody conjugate of the present disclosure is a tumor cell killing agent conjugated to an antibody via a thioether bond formed between a maleimide group possessed by the tumor cell killing agent of the third embodiment and a thiol group contained in an antibody having a thiol group. Hereinafter, the first tumor cell killing agent-antibody conjugate and the second tumor cell killing agent-antibody conjugate may be collectively referred to as the conjugate of the present disclosure. In the conjugate of the present disclosure, one molecule of the tumor cell killing agent may be conjugated to the antibody, or two or more molecules of the tumor cell killing agent may be conjugated to the antibody.

[0068] The antibody constituting the conjugate of the present disclosure is not particularly limited, but from the viewpoint of selectively accumulating the conjugate in tumor cells, anti-CD44 rat IgG (αCD44) is preferred, as it selectively binds to the CD44 molecule, a protein abundant on the surface of tumor cells. The method for conjugating the antibody to the tumor cell killing agent of the second embodiment is not particularly limited, and the method described in the section (Experimental Results 3) in the Examples can be used. The method for conjugating the antibody to the tumor cell killing agent of the third embodiment is not particularly limited, and a method of reacting the antibody with the tumor cell killing agent of the third embodiment under neutral conditions can be used.

[0069] (Application to Photoimmunotherapy (PIT)) The conjugate of the present disclosure may be applied to photoimmunotherapy (PIT) as follows. By administering the conjugate of the present disclosure to a subject, a tumor cell killing agent can be specifically accumulated in target cells or target tissues, such as tumor cells. By administering the conjugate of the present disclosure to a subject and irradiating the cells with excitation light in an amount effective to inhibit cell proliferation or induce cell death, cell proliferation inhibition or cell death can be induced, thereby treating the subject. Examples of subjects include humans and non-human mammals. Methods of administration to a subject include, but are not limited to, topical routes, injections (subcutaneous injection, intramuscular injection, intradermal injection, intraperitoneal injection, intratumoral injection, and intravenous injection), oral routes, ocular routes, sublingual routes, rectal routes, transdermal routes, intranasal routes, vaginal routes, and inhalation routes. The conjugate of the present disclosure may be administered alone, in the presence of a pharmaceutically acceptable carrier, or in the presence of other therapeutic agents. The conjugates of the present disclosure can bind to target cells or tissues, such as circulating tumor cells or cells of solid tumors. When irradiated with light, the tumor cell-killing agent constituting the conjugate of the present disclosure absorbs the light and generates singlet oxygen, which can damage or destroy the target cells or tissues. In photodynamic therapy, the wavelength of the irradiated light is preferably 660 nm to 850 nm, more preferably 700 nm to 850 nm. Light irradiation may be performed using a device equipped with a near-infrared (NIR) light-emitting diode. Light irradiation may be performed multiple times.

[0070] The present disclosure will be described in more detail below using examples, but the present disclosure is not limited to the following examples. <Preparation of Tumor Cell Killing Agent> [Synthesis of Compound 1] (Synthesis of Intermediate 1) To a suspension of 2,3-dicyanohydroquinone (3.42 g, 21.3 mmol) and potassium carbonate (8.82 g, 63.8 mmol) in dimethylformamide (40 mL) was added (2-bromoethoxy)(tert-butyl)dimethylsilane (11 mL, 51.6 mmol), and the mixture was heated and stirred at 50°C under a nitrogen atmosphere for 6 hours. Water was then added to the reaction solution, and the precipitated solid was filtered and washed with water. The solid was recrystallized from ethanol to obtain Intermediate 1 (6.82 g, 67%) represented by the following formula as a white solid. １ H-NMR (400MHz, CDCl ３ ) δ (ppm) 0.10 (s, 12H), 0.88 (s, 18H), 4.00 (t, 4H, J = 4.9Hz), 4.17 (t, 4H, J = 4.9Hz), 7.25 (s, 2H).

[0071]

[0072] (Synthesis of Compound 1-1) A suspension of intermediate 1 (7.00 g, 14.7 mmol) and diazabicycloundecene (2.21 mL, 14.7 mmol) in 1-pentanol (28 mL) was stirred at 125°C to dissolve intermediate 1. Palladium chloride (1.18 g, 6.67 mmol) was added to the solution, and the mixture was heated and stirred at 125°C to 130°C for 36 hours under a nitrogen atmosphere. The reaction solution was then filtered through Celite, and the filtrate was concentrated. The residue was purified by a silica gel column (toluene-ethyl acetate) and reprecipitation (dichloromethane-methanol) to obtain compound 1-1 (0.838 g, 11%) as a dark green solid. １ H-NMR (400MHz, CDCl ３ ) δ (ppm) 0.08 (s, 48H), 0.91 (s, 72H), 4.32 (t, 16H, J = 5.9Hz), 5.03 (t, 16H, J = 5.7Hz), 7.73 (s, 8H).

[0073]

[0074] (Synthesis of Compound 1) Triethylamine trihydrofluoride (2 mL, 12.3 mmol) was added to a solution of Compound 1-1 (0.838 g, 0.416 mmol) in tetrahydrofuran (4 mL), and the mixture was stirred at room temperature overnight. Thereafter, the solid precipitated from the reaction solution was filtered and washed with tetrahydrofuran to obtain Compound 1 (0.479 g, quant.) as a dark green solid. １ H-NMR (400MHz, d-DMSO) δ (ppm) 4.01-4.08 (m, 16H), 4.86 (t, 16H, J = 5.6Hz), 5.09 (t, 8H, J = 5.6Hz), 7.84 (s, 8H).

[0075]

[0076] [Synthesis of Compound 4] Succinic anhydride (0.043 g, 0.430 mmol) was added to a solution of Compound 1 (0.0467 g, 0.0450 mmol) and triethylamine (0.111 mL, 0.800 mmol) in dimethylformamide (1 mL), and the mixture was stirred at room temperature overnight. The reaction solution was then concentrated. The resulting residue was washed with tetrahydrofuran to give Compound 4 (0.0281 g, 35%) as a dark green solid. １ H-NMR (400MHz, d-DMF) δ (ppm) 2.50-2.58 (m, 16H), 2.60-2.67 (m, 16H), 4.81 (t, 16H, J = 5.2Hz), 5.26 (t, 16H, J = 4.8Hz), 7.90 (s, 8H).

[0077]

[0078] [Synthesis of Compound 2] Compound 4 (0.0758 g, 0.0399 mmol) was dissolved in 40 mM aqueous sodium hydroxide solution (8 mL, 0.32 mmol), and the water was then removed by lyophilization to obtain Compound 2 (0.0805 g, 97%) as a dark green solid.

[0079]

[0080] [Synthesis of Compound 3] (Synthesis of Intermediate 2) Using 2,3-dicyanohydroquinone (1.79 g, 11.2 mmol), potassium carbonate (5.44 g, 40.2 mmol), and 1-bromo-3-methoxypropane (3.26 mL, 29.2 mmol) as raw materials and reaction reagents, a white solid intermediate 2 (3.12 g, 92%) represented by the following formula was obtained in the same synthetic method as for Intermediate 1. １ H-NMR (400MHz, CDCl ３ ) δ (ppm) 2.05-2.12 (m, 4H), 3.36 (s, 6H), 3.58 (t, 4H, J=5,9Hz), 4.16 (t, 4H, J=6.2Hz), 7.19 (s, 2H).

[0081]

[0082] (Synthesis of Compound 3) Compound 3 (0.187 g, 17%) was obtained as a dark green oil by a synthetic method similar to that for Compound 1-1 using Intermediate 2 (1.02 g, 3.35 mmol), diazabicycloundecene (0.5 mL, 3.32 mmol), and palladium chloride (0.183 g, 1.03 mmol) as raw materials and reaction reagents. １ H-NMR (400MHz, CDCl ３ ) δ (ppm) 2.42-2.52 (m, 16H), 3.37 (s, 24H), 3.77 (t, 16H, J = 6.1Hz), 4.97 (t, 16H, J = 6.9Hz), 7.62 (s, 8H).

[0083]

[0084] [Synthesis of Compound 5] (Synthesis of Intermediate 3) Using 2,3-dicyanohydroquinone (3.05 g, 19.1 mmol), potassium carbonate (9.56 g, 69.2 mmol), and triethylene glycol-2-bromoethyl methyl ether (11 mL, 52.5 mmol) as raw materials and reaction reagents, a white solid intermediate 3 (6.67 g, 65%) represented by the following formula was obtained in the same synthetic method as for Intermediate 1. １ H-NMR (400MHz, CDCl ３) δ (ppm) 3.37 (s, 6H), 3.52-3.57 (m, 4H), 3.62-3.69 (m, 16H), 3.72-3. 76 (m, 4H), 3.89 (t, 4H, J=4.6Hz), 4.23 (t, 4H, J=4.6Hz), 7.25 (s, 2H).

[0085]

[0086] (Synthesis of Compound 5) Compound 5 (0.192 g, 6%) was obtained as a dark green oil by a synthetic method similar to that for Compound 1-1 using Intermediate 3 (2.82 g, 5.22 mmol), diazabicycloundecene (0.781 mL, 5.18 mmol), and palladium chloride (0.288 g, 1.63 mmol) as raw materials and reaction reagents. １ H-NMR (400MHz, CDCl ３ ) δ (ppm) 3.31 (s, 24H), 3.44-3.50 (m, 16H), 3.55-3.61 (m, 32H), 3.61-3. 68 (m, 32H), 4.19 (t, 16H, J=5.1Hz), 5.11 (t, 16H, J=5.1Hz), 7.7 (s, 8H).

[0087]

[0088] [Synthesis of Compound 6-1] (Synthesis of Intermediate 4) To a suspension of 2,3-dicyanohydroquinone (10.0 g, 62.7 mmol) and potassium carbonate (13.3 g, 96.1 mmol) in dimethylformamide (120 mL) was added (2-bromoethoxy)(tert-butyl)dimethylsilane (13.5 mL, 63.1 mmol), and the mixture was heated and stirred at 60°C under a nitrogen atmosphere for 11.5 hours. Water was then added to the reaction solution, and the organic layer was extracted with chloroform. The organic layer was dried over sodium sulfate, filtered, and concentrated to obtain a crude product. Iodomethane (3.0 mL, 48.2 mmol) was added to a suspension of the crude product and potassium carbonate (9.04 g, 65.4 mmol) in dimethylformamide (60 mL), and the mixture was stirred at room temperature for 18 hours under a nitrogen atmosphere. Water was then added to the reaction solution, and the organic layer was extracted with chloroform. The organic layer was dried over sodium sulfate, filtered, and concentrated. The residue was purified by a silica gel column (toluene-ethyl acetate) and recrystallization (ethanol) to obtain intermediate 4 (1.34 g, 6%) represented by the following formula as a white solid. １ H-NMR (400MHz, CDCl ３ ) δ (ppm) 0.10 (s, 6H), 0.89 (s, 9H), 3.93 (s, 3H) 4.00 (t, 2H, J = 5.4Hz), 4.17 (t, 2H, J = 4.9Hz), 7.15 (d, 1H, J = 9.5Hz), 7.29 (d, 1H, J=9.5Hz).

[0089]

[0090] (Synthesis of Compound 6-1) A dark green solid, Compound 6-1 (0.204 g, 6%), was obtained by a synthetic method similar to that for Compound 1-1 using Intermediate 4 (0.899 g, 2.70 mmol), 3,6-bis(2-ethoxyethoxy)1,2-benzene-dicarbonitrile) (2.48 g, 8.14 mmol), diazabicycloundecene (2.21 mL, 14.7 mmol), and palladium chloride (0.583 g, 3.29 mmol) as raw materials and reaction reagents. １ H-NMR (400MHz, CDCl ３) δ (ppm) 0.07 (s, 6H), 0.90 (s, 9H), 1.19-1.32 (m, 18H), 3.62-3.77 (m, 12H), 4.08-4.18 (m, 10H), 4.24 (t, 2H, J = 5.4Hz), 4.31 (t, 2H, J = 5.6Hz), 4.58 (s, 3H), 5.03-5.19 (m, 14H), 7.55 (d, 1H, J = 9.0Hz) 7.66-7.75 (m, H), 7.78 (d, 1H, J = 9.0Hz).

[0091]

[0092] [Synthesis of Compound 6-2] Compound 6-2 (0.0868 g, 38%) was obtained as a dark green solid by a synthetic method similar to that for Compound 1, using compound 6-1 (0.250 g, 0.185 mmol) and triethylamine trihydrofluoride (0.09 mL, 0.555 mmol) as raw materials and reaction reagents. １ H-NMR (400MHz, CDCl ３ ) δ (ppm) 1.17-1.32 (m, 18H), 3.60-3.78 (m, 12H), 3.99-4.07 (m, 2H), 4.08-4.16 (m, 8H), 4.18-4.27 (m, 4H), 4.59 (s, 3H), 4.82 (t, 2H, J = 4.1Hz), 5.04 (t, 2H, J = 5.4Hz), 5.08-5.18 (m, 10H), 5.40 (t, 1H, J = 6.7Hz), 7.59 (d, 1H, J = 8.8Hz), 7.67-7.77 (m, 7H).

[0093]

[0094] [Synthesis of Compound 6] Compound 6 (0.0257 g, 30%) was obtained as a dark green solid by a synthetic method similar to that for Compound 4 using compound 6-2 (0.0786 g, 0.0635 mmol), triethylamine (0.02 mL, 0.144 mmol), and succinic anhydride (0.0208 g, 0.208 mmol) as raw materials and reaction reagents. １H-NMR (400MHz, d-DMSO) δ (ppm) 1.21-1.29 (m, 18H), 2.45 (t, 2H, J = 6.7H z), 2.54 (t, 2H, J=7.2Hz), 3.63-3.75 (m, 12H), 4.12-4.15 (m, 10H), 4.2 2 (t, 2H, J = 5.6Hz), 4.56 (s, 3H), 4.73 (t, 2H, J = 4.6Hz), 5.09-5.14 (m, 1 2H), 5.24 (t, 2H, J = 10.0Hz), 7.53 (d, 1H, J = 8.5Hz), 7.68-7.71 (m, 7H).

[0095]

[0096] [Synthesis of Compound 7] Compound 7 (0.0678 g, 15%) was obtained as a dark green solid by a synthetic method similar to that for Compound 1-1 using 3,6-bis(2-ethoxyethoxy)phthalonitrile (0.429 g, 1.41 mmol), diazabicycloundecene (0.212 mL, 1.41 mmol), and palladium chloride (0.063 g, 0.355 mmol) as raw materials and reaction reagents. １ H-NMR (400MHz, CDCl ３ ) δ (ppm) 1.24 (t, 24H, J = 7.2Hz), 3.59-3.71 (m, 16H), 4.13 (t, 16H, J = 5.2Hz), 5.13 (t, 16H, J = 5.6Hz), 7.72 (s, 8H).

[0097]

[0098] Chlorin e6, represented by the following structure, was prepared as a comparative compound for compounds 1 to 7. Chlorin e6 has been widely studied as a commercially available singlet oxygen-generating dye (photosensitizer) and is approved by the U.S. Food and Drug Administration (FDA). Therefore, it was prepared as a representative example of a dye for photodynamic therapy (PDT) / photoimmunotherapy (PIT).

[0099]

[0100] <Solubility Test> Distilled water was added to a sample in a flask at 25°C, and the flask was shaken to stir. After stirring, it was visually confirmed whether the sample had dissolved. A sample was judged to be soluble in water when the concentration was equal to or higher than the concentration at which dissolution was confirmed visually. Compound 2 was used as a sample, and when it was dissolved in 1 g of water at 25°C, it was found that 10 mg or more dissolved. The solubility of Compound 2 in water at 25°C was 1 g / 100 g-H ２ It was O or above.

[0101] (Experimental Result 1) - Evaluation of Cytotoxicity (without Light Irradiation) - Compound 1 was dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution of 250 μM and 1 mM. Chlorin e6 (hereafter referred to as comparative compound 1) was prepared as a comparative example, and 250 μM and 1 mM stock solutions were prepared using the same procedure as for compound 1. Compound 2, which is highly water-soluble and poorly soluble in DMSO, was dissolved in 0.01 M phosphate-buffered saline (PBS) to prepare 250 μM and 1 mM stock solutions. Cultured tumor cells (mouse colon tumor cell line: Colon26) suspended in RPMI (Roswell Park Memorial Institute) 1640 medium were seeded into a 96-well plate at 5,000 cells per well and cultured at 37°C in a 5% CO2 incubator for 24 hours. Filter-sterilized 250 μM and 1 mM stock solutions of comparative compound 1, compound 1, and compound 2 were added to RPMI 1640 medium at 1 / 100 volumes to prepare final dilutions of 2.5 μM or 10 μM, respectively. To match the experimental conditions for compound 2 with other compounds, a group containing 1% DMSO (compound 2 + DMSO) was also prepared. Two control groups were prepared: medium containing no compound 1, compound 2, comparative compound 1, or solvent, and a 1% DMSO solution containing 1 / 100 volume of DMSO. After incubation, the culture medium was removed from the cells, and each diluted solution was added to the cells. The treated cells were cultured at 37°C in a 5% CO2 incubator for up to 7 days. WST8 assays were performed on cells on days 1, 2, 3, and 7 of incubation, and absorbance at 450 nm, indicating cell viability, was measured using a plate reader (BioTek Instruments). On the same day, microscopic observation was performed using a phase-contrast microscope (Carl Zeiss). Figure 1 shows the WST8 assay results, plotting the absorbance at 450 nm, which indicates cell viability, against the number of days since the start of culture. The graph on the left of Figure 1 shows the compound concentration at 2.5 μM, and the graph on the right shows the compound concentration at 10 μM.Plots show the average values ​​from three replicate experiments, and error bars indicate the standard deviation between the data. Statistical analysis using one-way analysis of variance and Dunnett's multiple comparison test (MM / SS) was used to calculate p values ​​indicating significant differences compared to the 1% DMSO-treated group. From day 1 to day 3 of culture, which corresponds to the logarithmic growth phase of tumor cells, no significant differences in viability were observed with Compound 1, Compound 2, or Comparative Compound 1. However, DMSO-induced growth inhibition was observed from day 3 onward. Days 3 to 7 of the figure correspond to the tumor cell death phase, during which viability declined in all groups, but no significant differences in viability were observed with Compound 1, Compound 2, or Comparative Compound 1. Figures 2 and 3 show phase-contrast images of cells taken with a phase-contrast microscope on day 1 (top) and day 3 (bottom). Low-magnification images (10x objective lens) in Figure 2 show no significant differences in cell density between the control group and the groups treated with Compound 1, Compound 2, or Compound 2 + DMSO at each concentration on day 1 and day 3 of treatment. On the other hand, no significant abnormalities in cell density were observed in the comparative compound 1-treated group at 2.5 μM, but the cells treated with 10 μM showed significantly lower cell density than the other groups on day 3 of treatment, suggesting cytotoxicity. Furthermore, comparison of the high-magnification images (40x objective lens) in Figure 3 revealed no significant differences in cell morphology among the groups. From the above examples, even at the 10 μM concentration where abnormalities were observed in the comparative compound 1-treated group, no significant cytotoxicity was observed in Compound 1 or Compound 2 itself, suggesting that these compounds are capable of coexisting with cells.

[0102] (Experimental Result 2) - Evaluation of Cytotoxicity (with Light Irradiation) - Comparative Compound 1 and Compound 1 were dissolved in DMSO to prepare stock solutions at concentrations of 250 μM and 1 mM. Compound 2 was dissolved in 0.01 M PBS to prepare stock solutions at 250 μM and 1 mM. Cultured tumor cells (mouse colon tumor cell line: Colon26) suspended in RPMI 1640 medium were seeded into a 96-well plate at 5,000 cells per well and cultured at 37°C in a 5% CO2 incubator for 24 hours. Filter-sterilized 250 μM stock solutions of Comparative Compound 1, Compound 1, and Compound 2 were added to RPMI 1640 medium at 1 / 100, 1 / 500, and 1 / 2,500 volumes to prepare final diluted solutions of 2.5 μM, 0.5 μM, and 0.1 μM, respectively. Furthermore, 10 μM diluted solutions were prepared by adding 1 / 100 of the 1 mM stock solutions of Comparative Compound 1, Compound 1, and Compound 2. To match the experimental conditions with other compounds, a group was also prepared for Compound 2 (Compound 2 + DMSO) by adding DMSO to the medium to achieve the respective solvent concentrations, as in (Experimental Results 1). As control groups, DMSO was diluted with RPMI 1640 medium to prepare 1%, 0.2%, and 0.04% DMSO diluted solutions according to the respective dilution amounts. After incubation, the culture medium was removed from the cells, and each solution was added to the cells. The treated cells were then cultured for 1 hour at 37°C in a 5% CO2 incubator. The group that underwent the next treatment in the diluted solution was designated the non-washed group, while the group that cultured the cells was removed from the diluted solution and re-added with RPMI 1640 medium alone was designated the washed group. To evaluate the cytotoxicity of compounds excited by light irradiation, two types of LED light sources (Select 100 LED Stand Light, Optocode Corporation) were prepared to match the maximum absorption wavelengths of comparative compound 1, compound 1, and compound 2. These light sources were arranged in an array with a central wavelength of 660 nm (maximum absorption wavelength of comparative compound 1) or 730 nm (maximum absorption wavelength of compounds 1 and 2).The group irradiated with an LED with a central wavelength of 660 nm was designated the 660 nm light irradiation group, and the group irradiated with an LED with a central wavelength of 730 nm was designated the 730 nm light irradiation group, with an output of 12 mW / cm for the non-washed and washed groups. 2 , energy amount 21.6 J / cm 2Light irradiation was performed under the following conditions. Cells were subjected to a WST8 assay 1 hour and 24 hours after light irradiation, and absorbance at 450 nm, indicating cell viability, was measured. On the same day, microscopic observations were performed using a phase-contrast microscope. Figure 4 shows microscopic images of cells irradiated with 660 nm light taken with a phase-contrast microscope (40x objective) 1 hour after irradiation. As shown in this figure, only the unwashed and washed groups treated with 10 μM Comparative Compound 1 exhibited swelling resembling cell death (necrosis). In contrast, no significant changes in cell morphology were observed in the other groups. Figure 5 shows microscopic images of cells irradiated with 730 nm light taken with a phase-contrast microscope (40x objective) 1 hour after irradiation. In this figure, images of cells exhibiting spherical morphology and swollen cells, indicating cell death, were observed in the unwashed and washed groups of 10 μM comparative compound 1, the unwashed group of 10 μM compound 1, and the unwashed groups of 2.5 μM and 10 μM compound 2 and compound 2 + DMSO. Figure 6, which shows cells 24 hours after 660 nm light irradiation, shows images of cells exhibiting spherical morphology and swollen cells, indicating cell death, in the unwashed groups of 2.5 μM and 10 μM comparative compound 1, the washed group of 10 μM comparative compound 1, and the unwashed group of 10 μM compound 2 and compound 2 + DMSO. Figure 7, which shows cells 24 hours after 730 nm light irradiation, shows images of cells exhibiting spherical morphology and swollen cells, indicating cell death, in the unwashed and washed groups of 10 μM comparative compound 1, and the unwashed groups of 2.5 μM and 10 μM compound 1 and compound 2 and compound 2 + DMSO. In addition, cells exhibiting a slight spherical morphology were observed in the 0.5 μM Compound 2 and Compound 2 + DMSO unwashed groups. No significant changes in cell morphology were observed in the control and 0.1 μM groups in Figures 4 to 6. The upper graphs in Figures 8 to 11 show the results of the WST8 assay, plotting the absorbance at 450 nm (representing cell viability) on the Y axis and the final concentration of each component on the X axis. The control DMSO concentrations plotted are 1% (10 μM and 2.5 μM control groups), 0.2%, and 0.04% for compound concentrations of 10 μM, 2.5 μM, 0.5 μM, and 0.1 μM, respectively.The lower panels of Figures 8 to 11 plot relative values, with the absorbance of the control group as the denominator and the absorbance of each treatment group as the numerator. The plots are the average values ​​of four replicate experiments, and error bars indicate the standard deviation between the data. Statistical analysis consisted of one-way analysis of variance and Dunnett's multiple comparison test. p values ​​were calculated to indicate significant differences relative to the 1% DMSO-treated group (top panels) and the comparative compound 1-treated group (bottom panels). Figure 8, which shows cell viability 1 hour after 660 nm light irradiation, reveals a significant decrease in cell viability only in the unwashed and washed groups treated with 10 μM comparative compound 1. Figure 9, which shows cell viability 1 hour after 730 nm light irradiation, reveals a tendency for decreased cell viability in the unwashed groups treated with 10 μM comparative compound 1, compound 1, compound 2, and compound 2 + DMSO, but no significant differences were detected. On the other hand, the relative value plots revealed that the washed group treated with 10 μM comparative compound 1 had significantly reduced cell viability compared to the compound 2 and compound 2 + DMSO groups. Figure 10, which shows cell viability 24 hours after 660 nm light irradiation, reveals a significant reduction in cell viability in the unwashed group treated with 2.5 μM comparative compound 1, compound 2, and compound 2 + DMSO, and the unwashed group treated with 10 μM comparative compound 1, compound 1, compound 2, and compound 2 + DMSO. Furthermore, the relative value plots revealed that compound 1 had significantly higher cell viability compared to the other treatment groups. On the other hand, in the washed group, only the 2.5 μM and 10 μM comparative compound 1 treatments showed a significant reduction in cell viability. Figure 11, which shows cell viability 24 hours after 730 nm light irradiation, reveals a significant reduction in cell viability in the unwashed group treated with 2.5 μM compound 2 and compound 2 + DMSO, and the unwashed group treated with 10 μM comparative compound 1, compound 1, compound 2, and compound 2 + DMSO. On the other hand, in the washed group, a significant decrease in cell viability was observed only with 10 μM of comparative compound 1. The results of phase contrast microscopy and the WST8 assay tended to generally agree, confirming that comparative compound 1, compound 1, and compound 2 exhibited significant cytotoxicity when irradiated with light at wavelengths corresponding to their respective maximum absorption wavelengths. Furthermore, the results of the WST8 assay suggest that the effective concentrations of each compound are estimated to be around 2.5 μM.Phase-contrast microscopy of the unwashed group of Compound 2 showed a slight induction of cell death at 0.5 μM. On the other hand, the WST8 assay results for Compound 1 showed no significant decrease in cell viability at 2.5 μM, suggesting its lower cytotoxicity compared to Comparative Compounds 1 and 2. Furthermore, Comparative Compounds 1, 1, and 2 were confirmed to exhibit significant cytotoxicity even when irradiated at wavelengths outside the maximum absorption wavelength (70 nm). The effective concentrations are estimated to be around 10 μM for Comparative Compounds 1 and 1, and around 2.5 μM for Compound 2, respectively. Since lower doses of pharmaceuticals are generally believed to reduce side effects, a lower effective concentration of Compound 2 than Comparative Compound 1 is useful. Since the cytotoxicity of Compounds 1 and 2 was observed only in the unwashed group, it is assumed that they do not permeate the cell membrane. However, since Compounds 1 and 2 are ultimately used in combination with antibodies, cell membrane permeability of Compounds 1 and 2 alone is not an issue. From the above examples, it can be seen that Compound 1 and Compound 2 are compounds that exhibit cytotoxicity upon light irradiation, and Compound 2 in particular has advantages over Comparative Compound 1, a current photosensitizing dye, in terms of effective concentration and effective wavelength range.

[0103] (Experimental Result 3) - Synthesis of Conjugates, Evaluation of Cytotoxicity and Cytotoxicity - Compound 2, which showed significant tumor cytotoxicity in (Experimental Result 2), was conjugated to an antibody to synthesize a conjugate, and the cytotoxicity and cytotoxicity of the conjugate were evaluated. Anti-CD44 rat IgG (αCD44), which recognizes the extracellular domain of the CD44 molecule, a cell surface antigen, was used as the antibody. An αCD44 isotype with no molecular recognition ability was also prepared as a control antibody. The conjugates were synthesized using the condensation agents 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide Hydrochloride (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS). Recipe 1: EDC, Sulfo-NHS, and compound 2 in a 1:1:1 molar ratio, antibody to compound 2 in a 1:25 molar ratio, and a 20-minute pretreatment. Recipe 2: antibody to compound 2 in a 1:200 molar ratio. Recipe 3: antibody to compound 2 in a 1:25 molar ratio without pretreatment. Recipe 4: antibody to compound 2 in a 1:200 molar ratio without pretreatment. The synthesis procedure involved dissolving EDC, Sulfo-NHS, and compound 2 in 50 mM MES buffer and pretreatment at room temperature to convert the carboxyl group of compound 2 into an active ester. The active ester and antibody were then reacted at room temperature for 24 hours to conjugate compound 2 to the amino group present in the lysine residues of the antibody. Note that recipes 3 and 4 involved direct reaction of EDC, Sulfo-NHS, compound 2, and antibody at room temperature for 24 hours without pretreatment. The reaction solution was subjected to solvent exchange with 0.01 M PBS using a 50 kDa cutoff ultrafiltration column, and the conjugate was concentrated. The absorbance of the concentrated conjugate at 730 nm derived from compound 2 was measured using a plate reader. Protein concentration was measured using the BCA (bicinchoninic acid) assay, and the molar ratio of antibody to compound 2 was calculated. The molar ratios of antibody to compound 2 were 0.4 (lot #1) for recipe 1, 18 (lot #2) for recipe 2, 7.6 (lot #3) for recipe 3, and 12.3 (lot #4) for recipe 4, respectively.The isotype was conjugated to Compound 2 according to Recipe 4, with a molar ratio of 9.8. The preparation of the conjugate is summarized in Table 1.

[0104]

[0105] For cell experiments, the following groups were prepared as controls: a 0.01 M PBS group (solvent); an αCD44 group (0.01 μM final concentration); a 10 μM Compound 2 group; an isotype group conjugated with Compound 2 at an antibody concentration of 0.01 μM (Isotype-Compound 2 group); and an αCD44 group conjugated with Compound 2 at an antibody concentration of 0.01 μM (αCD44-Compound 2 group, lots #1-4). To evaluate the toxicity of the compounds used for conjugation, a group was prepared in which the filtrate from the ultrafiltration column separated by Recipe 4 was treated at a concentration of 3% (3% v / v filtrate group). Another group was prepared in which the reaction solution of Recipe 4, excluding the antibody, was concentrated using the same procedure as for the conjugate and treated at a concentration of 0.3% (0.3% v / v conjugation reaction solution group). Cultured tumor cells (mouse colon tumor cell line: Colon26) suspended in RPMI1640 medium were seeded into a 96-well plate at 5,000 cells per well and cultured for 24 hours at 37°C in a 5% CO2 incubator. After culture, the culture medium was removed from the cells, and each of the treatment solutions was added to the cells. The cells were then cultured for 1 hour at 37°C in a 5% CO2 incubator. The non-washed group received the next treatment in the treatment solution, while the washed group received the treatment solution removed from the cultured cells and RPMI1640 medium alone. To evaluate the cytotoxicity of compounds excited by light irradiation, an LED light source (Select 100 LED Stand Light, Optocode Corporation) with a central wavelength of 730 nm, matching the maximum absorption wavelength of Compound 2, was prepared. The group exposed to LED light was designated the 730 nm light irradiation group, while the non-washed and washed groups received 12 mW / cm light. 2 , energy amount 21.6 J / cm 2Light irradiation was performed under the conditions of [0.01]. The group without light irradiation was designated the non-irradiated group. Cells 24 hours after light irradiation were subjected to a WST8 assay, and absorbance at 450 nm was measured to indicate cell viability. Simultaneously, microscopic observations were performed using a phase-contrast microscope at 1 and 24 hours. Figures 12 and 13 show microscopic images of cells in the non-irradiated group taken with a phase-contrast microscope (10x objective) at 1 and 24 hours, respectively. No significant abnormalities in cell density were observed in any of the treatment groups. On the other hand, Figures 14 and 15 show microscopic images of cells in the 730 nm light-irradiated group taken with a phase-contrast microscope (10x objective), respectively, at 1 and 24 hours. Figure 14, showing the results 1 hour after light irradiation, shows spherical morphology, indicating cell death, in the unwashed group containing 10 μM Compound 2 and 3% v / v filtrate. Furthermore, in the αCD44-Compound 2 Lots #2-4, most cells were observed to have a swollen morphology in both the washed and unwashed groups. A small number of cells with a spherical morphology, indicating cell death, were observed in Lot #1. Figure 15, showing the results 24 hours after light exposure, demonstrates that cells were swollen in the unwashed group containing 10 μM Compound 2 and 3% v / v filtrate, and in the washed and unwashed groups containing αCD44-Compound 2 Lots #2-4. As in Figure 14, a small number of cells with a spherical morphology, indicating cell death, were observed in Lot #1. The top panel of Figure 16 shows a graph of the WST8 assay, plotting the absorbance at 450 nm, which indicates cell viability, on the Y-axis. The bottom panel plots the relative values, with the absorbance of the control group as the denominator and the absorbance of each treatment group as the numerator. Figure 16 also lists the final concentrations of each component. The plots represent values ​​from a single experiment. The left panel of this figure shows cell viability after 24 hours in the non-irradiated group. Similar to Figure 13, no significant abnormalities in cell density were observed in any of the treatment groups. The right panel shows cell viability after 24 hours in the 730 nm light-irradiated group. Similar to Figure 15, significant decreases in cell viability were observed in the unwashed groups of the 10 μM Compound 2 and 3% v / v filtrate groups, and in the washed and unwashed groups of αCD44-Compound 2 Lots 2-4. There was also a slight tendency for cell viability to decrease in Lot 1.These examples demonstrate that αCD44-Compound 2 induces significant cytotoxicity upon photoirradiation. Based on the results for the αCD44 group and the conjugation reaction solution group, it was confirmed that this cytotoxicity was not due to the antibody itself or the compounds used in the conjugation reaction. Furthermore, while Compound 2 alone did not induce cytotoxicity in the washed group, αCD44-Compound 2 was confirmed to induce cell death even in the washed group. Furthermore, the αCD44-Compound 2 concentration was significantly lower at 0.01 μM compared to 10 μM for Compound 2 alone in the unwashed group, which showed cytotoxicity. ―３ The compound was effective at a concentration twice as high as the original compound. Its ability to function at low concentrations is advantageous as a pharmaceutical. Meanwhile, no cytotoxicity was observed with the isotype-compound 2, while αCD44-compound 2 is believed to fully maintain antibody function and bind to antigens on the tumor cell surface, suggesting tumor selectivity. Significant cytotoxicity was observed in lots #2-4, but only minimal cytotoxicity was observed in lot #1, suggesting that cytotoxicity is influenced by the molar ratio of antibody to dye. These findings demonstrate that compound 2 is a compound capable of binding to antibodies, and that its conjugate, αCD44-compound 2, exhibits significant cytotoxicity upon photoirradiation, demonstrating its potential for application in photoimmunotherapy.

[0106] (Experimental Result 4) - Evaluation of the Cell Death Induction Mechanism of Compound 2 - The cell death induction mechanism of Compound 2, which showed significant tumor cell cytotoxicity in (Experimental Result 2), was evaluated. In (Experimental Result 2), DMSO was added in addition to Compound 2. First, the interference of DMSO with cell death induction was evaluated. Compound 2 was dissolved in 0.01 M PBS to prepare a 1 mM stock solution. Cultured tumor cells (mouse colon tumor cell line: Colon26) suspended in RPMI 1640 medium were seeded into a 96-well plate at 5,000 cells per well and cultured at 37°C in a 5% CO2 incubator for 24 hours. A 1 / 100 volume of filter-sterilized Compound 2 1 mM stock solution was added to RPMI 1640 medium to prepare a diluted solution with a final concentration of 10 μM. DMSO was then added to this diluted solution to final concentrations of 0%, 0.5%, 1.0%, and 3.0%. After culturing, the culture medium was removed from the cells, and each solution was added to the cells. The cells treated as described above were cultured for 1 hour at 37°C in a 5% CO2 incubator. The group in which the next treatment was performed in the diluted solution was designated the non-washed group, and the group in which the diluted solution was removed from the cultured cells and only RPMI1640 medium was added again was designated the washed group. To evaluate the cytotoxicity of compounds excited by light irradiation, an LED light source with a central wavelength of 730 nm, which was matched to the maximum absorption wavelength of Compound 2, was used at an output of 12 mW / cm. 2 , energy amount 21.6 J / cm 2The cells were irradiated under the following conditions. A WST8 assay was performed on the cells 24 hours after light irradiation, and the absorbance at 450 nm, indicating cell viability, was measured. Cells 1 hour and 24 hours after light irradiation were also observed using a phase-contrast microscope. Figure 17 shows microscopic images of cells taken with a phase-contrast microscope (40x objective) 1 hour and 24 hours after light irradiation. In the unwashed group, cells exhibited spherical morphology and swollen cells, indicating cell death. In contrast, no significant changes in cell morphology were observed in the washed group. Figure 18 shows the results of a WST8 assay, plotting the absorbance at 450 nm, indicating cell viability, on the Y-axis and the final DMSO concentration on the X-axis. The plot represents the average of three replicate experiments, and the error bars indicate the standard deviation between the data. Statistical analysis included one-way analysis of variance and Dunnett's multiple comparison test. The p-values ​​for the upper panel indicated significant differences relative to the 0% DMSO-treated group. This figure shows no significant changes between all groups after 24 hours of light exposure. This suggests that DMSO does not interfere with the cell death induction of Compound 2. Next, the dependence of Compound 2's cell death induction on light exposure was evaluated. Stock solutions of Comparative Compound 1 and Compound 1 were prepared in DMSO at 1 mM. Compound 2 was dissolved in 0.01 M PBS to prepare a 1 mM stock solution. Cultured tumor cells (mouse colon tumor cell line: Colon26) suspended in RPMI1640 medium were seeded into a 96-well plate at 5,000 cells per well and cultured at 37°C in a 5% CO2 incubator for 24 hours. Filter-sterilized 1 mM stock solutions of Comparative Compound 1, Compound 1, and Compound 2 were added to RPMI 1640 medium at 1 / 100 volume to prepare diluted solutions with a final concentration of 10 μM. To match the experimental conditions with the other compounds, a group was also prepared for Compound 2 (Experimental Results 1), in which DMSO was added to the medium to a final concentration of 1% (Compound 2 + DMSO). As a control group, DMSO was diluted with RPMI 1640 medium to prepare a DMSO diluted solution with a final concentration of 1%. After culturing, the culture medium was removed from the cells, and each solution was added to the cells.The treated cells were cultured for 1 hour at 37°C in a 5% CO2 incubator. The non-washed group received the next treatment in the diluted solution, while the washed group received the diluted solution and RPMI 1640 medium. To evaluate the cytotoxicity of compounds excited by light, an LED light source with a central wavelength of 730 nm, matching the maximum absorption wavelength of Compound 2, was used at 4, 6, and 12 mW / cm. 2 The energy content was 21.6 J / cm at all power levels. 2 The cells were irradiated so that the light output was 4 mW / cm. 24 hours after the light irradiation, a WST8 assay was performed to obtain the absorbance at 450 nm, which indicates the cell viability. 24 hours after the light irradiation, the cells were also observed under a phase-contrast microscope. Figure 19 shows a microscopic image of the cells 24 hours after the light irradiation, taken with a phase-contrast microscope (40x objective). From this figure, it can be seen that the unwashed groups of Comparative Compound 1 and Compound 2 had the weakest output of 4 mW / cm. 2 Even at 6 mW / cm , images of spherical and swollen cells, indicating cell death, were observed. 2 In the washed group, only the comparative compound 1 was detected at an output of 6 mW / cm. 2 At this output, images of spherical cells and swollen cells, indicating cell death, were observed. The upper part of Figure 20 is a graph of the WST8 assay, with the Y-axis plotting the absorbance at 450 nm, which indicates cell viability, and the X-axis plotting the final concentration of each component. The lower part plots the relative values, with the absorbance of the control group (1% DMSO) as the denominator and the absorbance of each treatment group as the numerator. The plot is the average value of three repeated experiments, and the error bars indicate the standard deviation between the data. As with the phase-contrast microscope images, this figure shows that the weakest output of 4 mW / cm was used in the non-washed groups of Comparative Compound 1 and Compound 2. 2 On the other hand, a significant decrease in cell viability was observed in the non-washed group treated with Compound 1 at 4 mW / cm 2 Cell viability gradually decreased from 12 mW / cm 2In the washed group, only comparative compound 1 at 6 mW / cm 2 The results of phase contrast microscopy and WST8 assay tended to be generally consistent, with comparative compounds 1 and 2 exhibiting a tendency to decrease cell viability at an output of 4 mW / cm. 2The sensitivity was confirmed even at an output of 100 uA, demonstrating significant cytotoxicity. Next, to confirm whether the cell death induction of Compound 2 was due to singlet oxygen, cell death induction was evaluated in the presence of the antioxidant N-acetyl-L-cysteine ​​(NAC). Comparative Compound 1 and Compound 1 were dissolved in DMSO to prepare stock solutions at 1 mM. Compound 2 was dissolved in 0.01 M PBS to prepare a 1 mM stock solution. NAC was dissolved in 0.01 M PBS to prepare a 500 mM stock solution. Cultured tumor cells (mouse colon tumor cell line: Colon26) suspended in RPMI 1640 medium were seeded into a 96-well plate at 5,000 cells per well and cultured at 37°C in a 5% CO2 incubator for 24 hours. Filter-sterilized 1 mM stock solutions of Comparative Compound 1, Compound 1, and Compound 2 were added to RPMI 1640 medium at 1 / 100 volume to prepare dilutions with a final concentration of 10 μM. In addition, to match the experimental conditions with other compounds, a group containing Compound 2 (Compound 2 + DMSO) was also prepared. As a control group, DMSO was diluted with RPMI 1640 medium to prepare a 1% DMSO solution. Filter-sterilized 500 mM NAC was also added to RPMI 1640 medium to prepare diluted solutions with final concentrations of 10, 5, and 1 mM. After incubation, the culture medium was removed from the cells, and NAC solutions at the respective final concentrations were added to the cells. The cells were then cultured at 37°C in a 5% CO2 incubator for 1 hour. After 1 hour, without washing, comparative Compound 1, Compound 1, Compound 2, and DMSO diluted solutions were added, and the cells were then cultured at 37°C in a 5% CO2 incubator for 1 hour. To evaluate the cytotoxicity of compounds excited by light irradiation, an LED light source with a central wavelength of 730 nm, which was set to the maximum absorption wavelength of compound 2, was used at an output of 12 mW / cm. 2 , energy amount 21.6 J / cm 2The cells were irradiated without washing under the conditions of [0.001]. 24 hours after light irradiation, a WST8 assay was performed to measure the absorbance at 450 nm, indicating cell viability. Furthermore, the cells were observed using a phase-contrast microscope. Figure 21 shows microscopic images of the cells 24 hours after light irradiation taken with a phase-contrast microscope (40x objective). This figure shows that with Compound 1 and Compound 2, the number of spherical cells and swollen cells, indicating cell death, decreased in a concentration-dependent manner with the antioxidant NAC. In contrast, with Comparative Compound 1 and Compound 2 + DMSO, no decrease in cells indicating cell death was observed at any concentration of NAC. The top panel of Figure 22 shows a graph of the WST8 assay, plotting the absorbance at 450 nm, indicating cell viability, on the Y-axis and the final concentration of each component on the X-axis. The bottom panel plots the relative absorbance of each treatment group, with the absorbance of 0 mM NAC as the denominator and the absorbance of each treatment group as the numerator. The plots represent one experiment. Similar to the phase-contrast microscopy results, this figure demonstrates that compound 1 and compound 2 increased cell viability in a NAC concentration-dependent manner. On the other hand, a slight, but increasing, NAC concentration-dependent increase in cell viability was observed for comparative compound 1 and compound 2 + DMSO. The results of phase-contrast microscopy and WST8 assay generally agreed, demonstrating that cell death induction by compound 1 and compound 2 was suppressed by the antioxidant NAC. This suggests that cell death induction by compound 1 and compound 2 is mediated by singlet oxygen. On the other hand, cell death induction by comparative compound 1 was shown to be less sensitive to NAC. Comparative compound 1 (Experimental Result 2) is more cell-permeable, suggesting that singlet oxygen is produced intracellularly. In this study, weak intracellular internalization of NAC likely led to the reduced sensitivity of NAC. Furthermore, further verification of the sensitivity of NAC in compound 2 + DMSO is required. Based on these findings, compound 2 exhibited a 4 mW / cm 2 Even at this output, light irradiation induced significant cell death, and the mechanism was shown to be primarily singlet oxygen-induced cell death.

[0107] (Experimental Result 5) - Controlling the Labeling Rate of Antibody-Conjugates Conjugated with Compound 2 - In (Experimental Result 3), we prepared antibody-conjugated conjugates and confirmed their significant tumor cytotoxicity. In this study, we evaluated the control of the cytotoxicity of the conjugates by controlling the number of compound 2 molecules per antibody molecule (labeling rate). First, to identify synthesis conditions that would allow efficient control of the conjugate labeling rate, we used IgG-labeled agarose beads. These agarose beads were labeled with 1 mg of mouse IgG (antibody) per ml of suspension (0.068 nmol of IgG). First, we evaluated the ratio of EDC to compound 2 (EDC:Compound 2 ratio) and the pretreatment time (pre-reaction time) of EDC and Sulfo-NHS with compound 2 during the conjugate synthesis procedure. The synthesis procedure involved dissolving EDC, Sulfo-NHS, and compound 2 in 50 mM MES (2-morpholinoethanesulfonic acid) buffer and pre-treating at room temperature to convert the carboxyl group of compound 2 into an active ester. The active ester was then reacted with agarose beads at room temperature for 24 hours to conjugate compound 2 to the amino group present in the lysine residues of the antibody. After the reaction, the conjugate was recovered by centrifugation at 5000 x g for 1 minute. The absorbance of the conjugate at 730 nm derived from compound 2 was measured using a plate reader. Protein concentration was measured using the BCA assay, and the molar ratio of IgG to compound 2 was calculated to determine the conjugate labeling efficiency. Figure 23 shows the labeling efficiency of the conjugate obtained when controlling the pre-reaction time and EDC:compound 2 ratio. Note that the molar ratio of compound 2 to antibody used in this evaluation was 200:1 or 20:1. At both molar ratios of compound 2 to antibody, the longer the pre-reaction time, the lower the labeling rate, and the higher the EDC:compound 2 ratio, the higher the labeling rate. The highest labeling rate was achieved without pre-reaction time, but considering the possibility that EDC may also react with the carboxyl group of the antibody, a pre-reaction time of 5 minutes was deemed appropriate.On the other hand, the highest labeling rate was achieved at an EDC:compound 2 ratio of 8:1. However, excessive EDC may react with the carboxyl groups of the antibody. Since a 1:1 ratio also provided sufficient labeling, we determined that an EDC:compound 2 ratio of 1:0.5 or 1:1 was appropriate. Figure 24 shows a matrix plot of the labeling rate of the resulting conjugate when the ratio of compound 2 to antibody (compound 2:antibody ratio) was controlled. For this evaluation, we determined that an EDC:compound 2 ratio of 1:0.5 or 1:1 was appropriate based on Figure 23, so the EDC:compound 2 ratio was fixed at 1:0.5 and 1:1. The pre-reaction time was determined to be appropriate: 5 minutes, 0 minutes, and 20 minutes. Figure 24 demonstrates that increasing the compound 2:antibody ratio at 0 or 5 minutes of pre-reaction time increased the labeling rate. Furthermore, a 1:1 EDC:compound 2 ratio showed a high labeling rate. These evaluations suggest that the labeling rate of the conjugate can be controlled by adjusting the compound 2:antibody ratio during conjugate synthesis using a 1:1 EDC:compound 2 ratio and a 5-minute pre-reaction time. Next, the labeling rate of αCD44-compound 2 was evaluated under the labeling rate control conditions identified in the agarose bead evaluation. Furthermore, a conjugate of comparative compound 1 was also evaluated under the same conditions as the αCD44-compound 2 conjugate. The conjugate synthesis procedure involved dissolving EDC, sulfo-NHS, and compound 2 or comparative compound 1 in a 1:1:1 molar ratio in 50 mM MES buffer. A 5-minute pre-reaction time was performed at room temperature to convert the carboxyl group of compound 2 or comparative compound 1 into an active ester. The active ester was then reacted with αCD44 for 24 hours at room temperature to conjugate compound 2 or comparative compound 1 to the amino group present in the lysine residues of αCD44. The reaction solution was subjected to solvent exchange with 0.01 M PBS using a 50 kDa cutoff ultrafiltration column, and the conjugate was concentrated. Figure 25 shows the absorption spectra of αCD44 or Compound 2, Comparative Compound 1, αCD44-Compound 2, and αCD44-Comparative Compound 1. From this figure, it is clear that each conjugate exhibits a compound-specific absorption spectrum, suggesting that each compound is conjugated to αCD44.Figure 26 shows the labeling efficiency of αCD44-Compound 2 or αCD44-Comparative Compound 1 obtained when controlling the ratio of Compound 2:antibody or the ratio of Comparative Compound 1:antibody. The horizontal axis represents the number of compounds per antibody at the time of reaction setup, and the vertical axis represents the labeling efficiency, which represents the number of compounds bound to one antibody in the conjugate. This figure shows a linear correlation between the ratio of Compound 2:antibody and the labeling efficiency, R. 2 On the other hand, the comparative compound 1:antibody ratio and labeling rate show a linear correlation up to a labeling rate of about 2, but at labeling rates above that, the deviation becomes high and R 2The labeling efficiency was 0.465. Furthermore, it was confirmed that compound 2, compared with comparative compound 1, achieved a high labeling efficiency with fewer molecules. Figure 27 shows the estimated labeling conditions for a desired labeling efficiency based on the plot of compound:antibody ratio and labeling efficiency obtained from Figure 26. Because compound 2 can stably label even at high conjugate labeling efficiency, labeling conditions (Low (L), Middle (M), and High (H)) were determined to achieve labeling efficiency of 2, 4, and 6. On the other hand, comparative compound 1 tends to exhibit high deviations above a labeling efficiency of 2, so labeling conditions (Low (L), Middle (M), and High (H)) were determined to achieve labeling efficiency of 1, 2, and 3. The labeling efficiency of each conjugate conjugated under these conditions was plotted in a graph. This figure confirmed that it was possible to obtain conjugates with desired labeling efficiency under each condition. Next, we evaluated whether the conjugates conjugated with compound 2 retained the antigen recognition function inherent to antibodies. First, a conjugate with a labeling ratio of approximately 2 was synthesized under low (L) labeling conditions. Then, a conjugate with a labeling ratio of approximately 9 was synthesized under higher labeling conditions. The maintenance of antibody structure in these conjugates was assessed by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The conjugate, αCD44, and compound 2 were mixed in 4x SDS sample buffer (240 mM Tris-HCl (pH 6.8), 8% SDS, 40% glycerol, and 0.1% BPB (bromophenol blue)) to a final concentration of 0.2 mg / ml for the conjugate and αCD44. Compound 2 was similarly adjusted to final concentrations of 1, 10, and 100 μM. Furthermore, samples were prepared by mixing αCD44 and compound 2 without conjugation, with final concentrations of 0.2 mg / ml and 100 μM for each. 2-Mercaptoethanol was added to these samples to a final concentration of 5%, and the final samples were prepared by boiling at 95°C for 5 minutes. 10 μL of each sample was loaded onto a 15% SDS PAGE gel and electrophoresis was performed at a constant current of 20 mA.Figure 28 shows an image of the gel after electrophoresis stained with CBB (Coomassie Brilliant Blue) stain. Antibody molecules consist of two heavy and two light chains. Marker sample (L) showed clear bands at 50 kDa and 25 kDa, representing the heavy and light chains, for αCD44. On the other hand, heavy and light chain bands were also observed for αCD44-Compound 2. Furthermore, due to the increased molecular weight of each molecule due to the conjugation of Compound 2, the bands of αCD44-Compound 2 were slightly upshifted relative to the band of αCD44. The lower intensity of the band of αCD44-Compound 2 relative to the band of αCD44 is presumably due to steric or electrostatic inhibition of the interaction of CBB molecules by the conjugation of Compound 2. Furthermore, no band of Compound 2 was observed in the band of αCD44-Compound 2. Therefore, the content of free Compound 2 in the αCD44-Compound 2 solution is considered to be significantly low. On the other hand, in the αCD44-Compound 2 band, in addition to the heavy and light chain bands, a band was observed at the high molecular weight side. This band, which is thought to represent an antibody-antibody complex, was not observed with αCD44, but a similar band was observed in a sample in which αCD44 and Compound 2 were mixed without adding an active esterifying agent. Therefore, this band, which is thought to represent a complex, is not due to a conjugation reaction between carboxyl and amino groups, but rather is a band formed by antibody-antibody conjugation due to reactive oxygen species generated by Compound 2. Next, Western blotting was performed to evaluate the antigen recognition ability of the conjugate. Colon 26 homogenate was prepared as the antigen sample, and further, a sample for SDS-PAGE was prepared using the above method. This sample was loaded onto a 7.5% SDS-PAGE gel so that the protein amounts were 1.5, 1, and 0.5 mg, and electrophoresis was performed in constant current mode at 20 mA. After electrophoresis, proteins were transferred from the gel to a PVDF (polyvinylidene fluoride) membrane, and αCD44 adjusted to a final concentration of 7.5 nM and αCD44-Compound 2 with labeling ratios of approximately 2 and 9 were added to the membrane to perform the antibody-antigen reaction.The bands were then visualized by adding a horseradish peroxidase (HRP)-conjugated secondary antibody and chemiluminescence. Figure 29 shows the image after the chemiluminescence reaction. From this figure, it was observed that the band pattern of αCD44-Compound 2 at labeling ratios of approximately 2 and 9 was similar to that of αCD44. Note that multiple variant forms of the antigenic CD44 molecule exist, resulting in multiple bands. Furthermore, to evaluate whether antigen recognition was observed not only in the homogenate but also in the cells, immunostaining of cells was performed. Cultured tumor cells (mouse colon tumor cell line: Colon26) suspended in RPMI 1640 medium were seeded into 8-well glass-bottom chambers at 10,000 cells per well and cultured at 37°C in a 5% CO2 incubator for 24 hours. After culturing, the culture medium was removed from the cells, and a mixture of the isotype, αCD44, and αCD44-Compound 2 with labeling ratios of approximately 2 and 9 was added to RPMI 1640 medium to a final concentration of 60 nM. The treated cells were cultured for 1 hour at 37°C in a 5% CO2 incubator. After 1 hour, the cells were washed with 0.01 M PBS and then chemically fixed with 4% PFA (paraformaldehyde). αCD44 was then visualized by fluorescence using a fluorescently labeled secondary antibody solution and mounted in a mounting medium along with Hoechst 33342, a fluorescent DNA stain. Figure 30 shows confocal microscopy images of the samples prepared by this process. This figure shows that αCD44-Compound 2 with labeling ratios of approximately 2 and 9 exhibited a fluorescent signal pattern similar to that of αCD44, suggesting that the compound 2 recognized the antigen on the cells. These results suggest that the labeling ratio can be controlled in the synthesis of conjugates using Compound 2. Furthermore, it is possible to synthesize a conjugate with a higher labeling rate than with comparative compound 1. This is thought to be because the excellent hydrophilicity of compound 2 allows the synthesis of a conjugate without reducing the hydrophilicity of the antibody.Furthermore, the synthesized conjugates retained the antibody structure and function at least up to a labeling ratio of approximately 9, enabling normal recognition of antigens in cell homogenates and cells. Furthermore, Figure 31 shows Western blotting images of αCD44-Compound 2 and αCD44-Comparative Compound 1 synthesized under the labeling conditions of Low (L), Middle (M), and High (H) set in Figure 27. This image was obtained using the same procedure as Figure 29. As can be seen from this image, αCD44-Compound 2 exhibited a band pattern similar to αCD44 under all labeling conditions, whereas αCD44-Comparative Compound 1 exhibited a stronger signal than αCD44 and distinct bands were also observed. This suggests that αCD44-Compound 2 retains its antigen recognition ability compared to αCD44-Comparative Compound 1.

[0108] (Experimental Result 6) - Evaluation of cytotoxicity of conjugates with controlled labeling rates - (Experimental Result 5) confirmed that the labeling rate can be controlled in the synthesis of conjugates using Compound 2 and that these conjugates retain their functionality. In this study, we evaluated the cytotoxicity of conjugates with controlled labeling rates. Cultured tumor cells (mouse colon tumor cell line: Colon 26) suspended in RPMI 1640 medium were seeded into a 96-well plate at 5,000 cells per well and cultured at 37°C in a 5% CO2 incubator for 24 hours. αCD44-Compound 2 or αCD44-Comparative Compound 1 synthesized under low (L), middle (M), or high (H) labeling conditions were diluted in RPMI 1640 medium to final concentrations of 1, 5, and 10 nM. As controls, diluted solutions of αCD44 (final concentration: 10 nM, corresponding to the maximum antibody concentration of the conjugate), and Compound 2 and Comparative Compound 1 (final concentrations: average 53 and 42 nM, respectively, corresponding to the maximum compound concentration of the conjugate) were prepared. As controls, diluted solutions were prepared by adding 0.01 M PBS, the conjugate solvent, to the maximum volume at the time of sample addition. After incubation, the culture medium was removed from the cells, and each solution was added to the cells. The treated cells were cultured for 1 hour at 37°C in a 5% CO2 incubator. The non-washed group was treated in the diluted solution, while the washed group was cultured in the diluted solution, where the diluted solution was removed and RPMI 1640 medium alone was added again. To evaluate the cytotoxicity of compounds excited by light irradiation, an LED light source with a central wavelength of 660 nm (maximum absorption wavelength of Comparative Compound 1) or 730 nm (maximum absorption wavelength of Compound 2) was used, with an output of 12 mW / cm. 2 , energy amount 21.6 J / cm 2Cells were irradiated under the following conditions. 24 hours after light irradiation, a WST8 assay was performed to measure the absorbance at 450 nm, indicating cell viability. Furthermore, cells 24 hours after light irradiation were observed using a phase-contrast microscope. Figure 32 shows micrographs of cells in the unwashed group of αCD44-Compound 2-treated cells, taken with a phase-contrast microscope (10x objective) 24 hours after irradiation with 730 nm light. This figure shows a decrease in cell number at 10 nM and 5 nM in labeling conditions M and H, respectively. No significant changes in cell number were observed in the other groups. Figure 33 shows micrographs of cells in the washed group of αCD44-Compound 2-treated cells, taken with a phase-contrast microscope (10x objective) 24 hours after irradiation with 730 nm light. This figure shows a decrease in cell number at 5 nM in both labeling conditions M and H. No significant changes in cell number were observed in the other groups. Figure 34 shows micrographs of cells in the unwashed group of αCD44-comparative compound 1-treated cells, taken with a phase-contrast microscope (10x objective) 24 hours after irradiation with 660 nm light. No significant changes in cell number were observed in any of the groups. Figure 35 shows micrographs of cells in the washed group of αCD44-comparative compound 1-treated cells, taken with a phase-contrast microscope (10x objective) 24 hours after irradiation with 660 nm light. No significant changes in cell number were observed in any of the groups. Figure 36 shows micrographs of cells in the unwashed group of αCD44-comparative compound 2-treated cells, taken with a phase-contrast microscope (40x objective) 24 hours after irradiation with 730 nm light. In the labeling conditions M and H, spherical and swollen cells, indicating cell death, were observed at 10 nM and 5 nM, respectively. However, no significant changes in cell morphology were observed in the other groups. Figure 37 shows phase-contrast images (40x objective) of cells in the washed group containing αCD44-Compound 2, taken 24 hours after irradiation with 730 nm light. Images of spherical and swollen cells, indicative of cell death, were observed at 5 nM and 10 nM in labeling conditions M and H or L, respectively. No significant changes in cell morphology were observed in the other groups.Figure 38 shows micrographs of cells from the unwashed group of cells treated with αCD44-comparative compound 1, taken with a phase-contrast microscope (40x objective) 24 hours after irradiation with 660 nm light. No significant changes in cell morphology were observed in any of the groups. Figure 39 shows micrographs of cells from the washed group of cells treated with αCD44-comparative compound 1, taken with a phase-contrast microscope (40x objective) 24 hours after irradiation with 660 nm light. No significant changes in cell morphology were observed in any of the groups. The top panel of Figure 40 shows a graph of the WST8 assay, plotting the absorbance at 450 nm (representing cell viability) on the Y-axis and the final concentration of each component on the X-axis. The bottom panel plots the relative absorbance of each treatment group, with the absorbance of the PBS control group as the denominator and the absorbance of each treatment group as the numerator. The plots represent the average of two replicate experiments, and the error bars indicate the standard deviation between the data. This figure shows that for αCD44-Compound 2, a significant decrease in cell viability was observed in a concentration-dependent manner starting from 5 nM under labeling conditions M and H. On the other hand, under labeling condition L, a tendency for cell viability to decrease was observed, although the decrease was higher than in M ​​and L. Furthermore, cell viability tended to be lower in the washed group than in the unwashed group. For αCD44-Comparative Compound 1, no significant decrease in cell viability was observed in any of the groups. Next, the concentration range that showed significant cell viability under labeling condition L, which has the lowest labeling rate, was evaluated. Using the same procedure as described above, diluted solutions of αCD44-Compound 2 or αCD44-Comparative Compound 1 synthesized under labeling condition L were evaluated at final concentrations of 1, 5, 10, 50, and 100 nM. Figure 41 shows the results of a WST8 assay, plotting the absorbance at 450 nm (representing cell viability) on the Y axis and the final concentration of each component on the X axis. The lower panel plots the relative absorbance values, with the PBS control as the denominator and the absorbance of each treatment group as the numerator. The plots represent the average values ​​of two replicate experiments at 50 and 100 nM. The error bars indicate the standard deviation between the data. As shown in this figure, αCD44-Compound 2 synthesized under labeling condition L showed a significant decrease in cell viability from 50 nM. Furthermore, as with the conjugates under other labeling conditions, cell viability tended to be lower in the washed group than in the unwashed group.On the other hand, no significant decrease in cell viability was observed in any of the αCD44-comparison compound 1 groups. These results demonstrate that when αCD44 and a dye are conjugated, Compound 2 exhibits superior cytotoxicity compared to Comparison Compound 1. Furthermore, the higher the conjugate's labeling efficiency, the lower the cytotoxicity. This conjugate exhibits cytotoxicity at lower concentrations than Compound 2 alone. This is presumably due to the effect of αCD44, which generates singlet oxygen when the conjugate binds to cells, efficiently inducing cell death. Furthermore, the tendency for cell viability to be lower in the washed group than in the unwashed group is presumably due to the adverse effect of singlet oxygen generated by the free conjugate on the antigen-recognized conjugate.

[0109] (Experimental Result 7) - Evaluation of the antitumor effect of the conjugate - In (Experimental Result 6), it was confirmed that the conjugate with controlled labeling rate exhibited cytotoxicity. In this test, the conjugate under labeling condition H, which showed the highest cytotoxicity, was used to evaluate its antitumor effect on tumor-inoculated animals. In this test, 5x10 cultured tumor cells (mouse colon tumor cell line: Colon26) were placed in a solution made by mixing HBSS (Hank's Balanced Salt Solution) and Matrigel at a ratio of 2:1. ５ A cell suspension was prepared at a concentration of 100 cells / mL. 100 μL of this cell suspension was subcutaneously transplanted into both dorsal flanks of Balb / cA mice, the same species as Colon26, under anesthesia. After transplantation, body weight and tumor diameter were measured twice a week. For tumor diameter measurement, the long and short diameters of the tumor were measured, and the formula was (long diameter / 2) x (short diameter). 2 In this study, tumor volumes were calculated using the following formula: 3Mice reaching this level were used as tumor-implanted animals for the experiment. First, in this study, we evaluated the biotoxicity of the conjugates synthesized under labeling condition H. Figure 42 shows the experimental groups used in this study. Furthermore, for the synthesis of the conjugates administered to tumor-implanted animals, rather than the small-scale synthesis described above, we performed synthesis under scaled-up conditions appropriate for the dosage. This figure shows the labeling yields of the conjugates synthesized under labeling condition H at both a small scale (2 nmol antibody) and an expanded scale (50 nmol antibody). Note that in the expanded scale, the ratios were kept the same, but the volumes of other reagents were increased to match the antibody concentration. This figure demonstrates that under labeling condition H, the labeling yield improved with increasing synthesis scale. (Experimental Results 4) showed that antibody function was maintained at a labeling yield of approximately 9, so we used the conjugates synthesized under this scale. All of the administered reagents shown in this figure were administered systemically via the tail vein at a dose of 100 μl / 25 g. This figure shows the weight changes over time in animals administered the conjugates under labeling condition H, αCD44, and PBS as a control. No rapid weight loss was observed in the short term after administration of each reagent. Furthermore, 24 hours after administration, animals were euthanized, and tissues were excised and chemically fixed in 4% PFA. The fixed specimens were then sliced ​​at 10 μm thickness using a cryostat. Figure 43 shows micrographs of liver and kidney sections stained with hematoxylin and eosin (H&E) and captured under a phase-contrast microscope. No significant tissue lesions or damage were observed in the liver and kidney, which are important drug-metabolizing organs, in any of the groups. Next, αCD44 was visualized in tumor sections using a fluorescently labeled secondary antibody solution and mounted in a mounting medium with Hoechst 33342, a fluorescent DNA stain. Figure 44 shows confocal microscopy images (10x objective) of the samples prepared by the above treatment. From this figure, fluorescent signals were observed for both αCD44 and αCD44-Compound 2, indicating that the administered αCD44 was present within the tumor. The fluorescent signals tended to be strong around blood vessels, and the signal was stronger for αCD44-Compound 2 than for αCD44.Figure 45 shows confocal microscopy images (20x objective) of the sample prepared by the above treatment. This figure demonstrates cell membrane-like fluorescent signals for both αCD44 and αCD44-Compound 2. Furthermore, as in Figure 44, a stronger signal was observed for αCD44-Compound 2 than for αCD44. These results confirm that the conjugate of Compound 2 conjugated to αCD44 does not exhibit significant acute toxicity in vivo and accumulates to a certain extent in tumors. Next, tumor-inoculated animals were administered the conjugate, and the antitumor effect of laser irradiation was evaluated. Figure 46 shows the experimental groups used in this study. In the light-irradiated group, laser irradiation was performed only on the right tumor, while the left tumor served as a control without laser irradiation. Figure 47 shows the actual light irradiation conditions used in this study. In this study, a 730 nm laser diode was used to irradiate the tumor via a multimode optical fiber. The optical fiber used could be branched into two optical fibers by fusion splicing. Therefore, simultaneous laser irradiation of two animals is possible. Figure 48 shows the progression of tumor volume in the experimental groups shown in Figure 46. This graph plots tumor volume on the Y axis and the number of days since the day of each reagent administration (day 0) on the X axis. Figure 49 plots the relative values, with the tumor volume on each reagent administration day as the denominator and the tumor volume from the following day onwards as the numerator, and the number of days since the day of each reagent administration as the X axis. In these figures, Rat IgG is the control group for the effects of drug administration, αCD44 is the control group for the effects of antibody, and αCD44-Compound 2 is the control group for the effects of the conjugate itself. For the right-sided tumor, the laser irradiation experimental group (15 mg / kg αCD44-Compound 2 (2LD, 100J) _ 24 hpi) demonstrated significant tumor growth suppression compared to the control groups. Furthermore, a comparison of the antitumor effects between 15 mg / kg and 5 mg / kg administration doses revealed no significant differences in this study. Furthermore, a comparison of the antitumor effects of different laser irradiation energy densities was performed, and the results were 30 J / cm. 2 More than 100 J / cm 2The tumor growth inhibitory effect was confirmed at 24 hours after administration. Furthermore, when comparing a group that received a single laser irradiation 24 hours after administration with a group that received two laser irradiations, 24 and 48 hours after administration, the group that received two laser irradiations tended to have a higher tumor growth inhibitory effect. Figure 50 shows the results of a comparison of the antitumor effects observed between the 15 mg / kg αCD44-Compound 2 (2LD, 100J)_1hpi group, in which laser irradiation was performed 1 hour and 24 hours after administration of the reagent, and the 15 mg / kg αCD44-Compound 2 (2LD, 100J)_24hpi group, in which laser irradiation was performed 24 and 48 hours after administration of the reagent. In addition to the control group, a group 2LD was prepared in which laser irradiation alone was performed without administering the reagent to examine the effect of laser irradiation on the tumor. This figure confirms that 15 mg / kg αCD44-Compound 2 (2LD, 100J)_1hpi has a greater tumor growth inhibitory effect than 15 mg / kg αCD44-Compound 2 (2LD, 100J)_24hpi. Figure 51 shows representative photographs of tumors in 15 mg / kg αCD44-Compound 2 (2LD, 100J)_1hpi and in Group 2LD, which was treated with laser irradiation only without reagent administration. This figure shows that tumors in 15 mg / kg αCD44-Compound 2 (2LD, 100J)_1hpi were smaller than those in Group 2LD, demonstrating a significant tumor-inhibitory effect. These results suggest that αCD44-Compound 2 exhibits an antitumor effect upon laser irradiation in tumor-inoculated animals, particularly at an irradiation energy density of 100 J / cm. 2 48-50, a high antitumor effect was observed when laser irradiation was performed twice, 1 hour and 24 hours after administration. Also, as shown in Figures 48-50, even in the left tumor that was not irradiated with laser, an administration volume of 15 mg / kg resulted in 100 J / cm 2In the group in which the right tumor was irradiated twice with a laser with an energy density of 1000 kJ / s, the growth of the left tumor tended to be suppressed regardless of the timing of the laser irradiation. Therefore, it is speculated that the killing of the right tumor by laser irradiation may have stimulated the immune system, leading to the treatment of the left tumor. Figure 52 shows autopsy photographs taken after the first laser irradiation in the group in which the first laser irradiation was administered 1 hour after administration. This figure shows that tissue oxidation was observed in the liver in the group in which the first laser irradiation was administered 1 hour after administration. This suggests that αCD44-Compound 2 also accumulated in the liver 1 hour after administration. Furthermore, it is thought that the irradiated laser reached the liver deeper than the tumor, thereby exciting αCD44-Compound 2, which accumulates in the liver. Therefore, αCD44-Compound 2 and Compound 2 can be excited even in very deep tissues in the body and induce tissue oxidation. However, this tissue oxidation in the liver was almost completely resolved 2 weeks after laser irradiation. Furthermore, this study evaluated whether tumor cell death within the tumor was induced by laser irradiation. In the 15 mg / kg αCD44-Compound 2 (2LD, 100J)_1hpi group, which showed the highest antitumor effect, tumors were excised 24 hours after the second laser irradiation. The excised tumors were chemically fixed with 4% PFA. The fixed specimens were then sliced ​​at 10 μm thickness using a cryostat. Figures 53 and 54 are micrographs of the sliced ​​specimens stained with H&E and captured using a phase-contrast microscope. Figure 53 shows that the tumor on the non-laser-irradiated side (left side) was densely populated with tumor cells on the surface and center of the tumor. In contrast, the tumor on the laser-irradiated side (right side) was sparsely populated in the area indicated by the white line in Figure 54. Observation using a 40x objective lens revealed tumor cell death in this area, suggesting it was a necrotic region. Furthermore, numerous dilated blood vessels were observed within the tumor on the laser-irradiated side (right side), as indicated by the arrows in Figure 54. From the above, this test confirmed that the conjugate with controlled labeling rate exhibited antitumor effects in tumor-implanted animals.In this study, the dose was 15 mg / kg, and the intensity was 100 J / cm 1 and 24 hours after administration. 2 It was found that the highest antitumor effect was observed when the laser was irradiated twice at 1000 nm. In this group, observation of tumor cells within the tumor revealed that the tumor cells had undergone cell death, resulting in necrosis within the tumor.

[0110] (Experimental Result 8) - Evaluation of the pharmacokinetics of the conjugate - (Experimental Result 7) confirmed that the conjugate with a controlled labeling rate exhibits cytotoxicity in vivo, thereby demonstrating antitumor effects. On the other hand, (Experimental Result 7) showed that in this test, differences in antitumor effects were observed between the groups in which the first laser irradiation was performed 1 hour and 24 hours after administration. Therefore, in this test, the pharmacokinetics of the conjugate was evaluated. In this test, 5x10 cultured tumor cells (mouse colon tumor cell line: Colon26) were placed in a solution containing a 2:1 mixture of HBSS and Matrigel. ５ A cell suspension was prepared at a concentration of 100 cells / mL. 100 μL of this cell suspension was subcutaneously transplanted into both dorsal flanks of Balb / cA mice, the same species as Colon26, under anesthesia. After transplantation, body weight and tumor diameter were measured twice a week. For tumor diameter measurement, the long and short diameters of the tumor were measured, and the formula was (long diameter / 2) x (short diameter). 2 In this study, tumor volumes were calculated using the following formula: 3Mice reaching this age were used as tumor-implanted animals for this experiment. In this study, the pharmacokinetics of conjugates labeled under conditions H and L was evaluated. Each conjugate was administered systemically via the tail vein at a dose of 100 μl / 25 g. Animals were euthanized 1 and 24 hours after administration, and the kidneys, liver, and tumors were removed and chemically fixed in 4% PFA. Fixed specimens were then sliced ​​at 10 μm thickness using a cryostat. αCD44 was visualized in the sections using a fluorescently labeled secondary antibody solution and mounted in mounting medium with Hoechst 33342, a fluorescent DNA stain. The samples were imaged using a confocal microscope (2.5x objective lens), and the fluorescence intensity of αCD44 was analyzed in the resulting images. Analysis was performed using ImageJ image analysis software. Three 50-pixel square regions were randomly selected from each tissue image, and the sum of αCD44 fluorescence intensities within each region was measured. The average of the three sums of fluorescence intensities is shown in Figure 55. The plot is relative to the value obtained from the tissues of animals treated with PBS as the control. In the kidney, the group treated with the H conjugate 1 hour after administration showed the highest fluorescence intensity, while the other groups showed comparable values. In the liver, the group treated with the L conjugate 1 hour after administration showed the highest fluorescence intensity, while the other groups showed comparable values. In the tumor, the group treated with the H and L conjugates 1 hour after administration showed the highest fluorescence intensity, while the groups treated with the H and L conjugates 24 hours after administration showed comparable values. Analysis limited to the tumor surface showed no significant differences between the groups. In contrast, analysis limited to the tumor center showed the group treated with the H and L conjugates 1 hour after administration showed the highest fluorescence intensity, while the groups treated with the L conjugates 24 hours after administration showed comparable values. It is well known that administered drugs are metabolized according to their hydrophobicity and molecular weight. Highly hydrophobic drugs are metabolized primarily in the liver, while less hydrophobic drugs are metabolized primarily in the kidneys. From this study, compound 2 is highly water-soluble, and it is assumed that under labeling condition H, which has a higher labeling rate, metabolism in the kidneys is predominant. On the other hand, under labeling condition L, which has a lower labeling rate, metabolism in the liver is predominant.Furthermore, both conjugates reached their peak metabolic activity within 24 hours of administration, with very low levels of metabolism observed after 24 hours. In contrast, tumors showed peak accumulation within 1 hour of administration, suggesting that conjugate degradation or diffusion within the tissue may have begun within 24 hours. If the conjugate diffuses within the tissue, the fluorescence intensity becomes very weak, making it difficult to detect using this analysis. Furthermore, higher accumulation was observed in the tumor center than the surface. Higher blood leakage in the tumor center is likely responsible for enhanced accumulation. Figure 56 shows confocal microscopy images taken with a 10x objective lens, revealing discrete high-intensity signals in the tumor tissue. Figure 57 shows tumor tissue from the group administered with the conjugate under labeling condition H, which showed the highest signal intensity in the analysis shown in Figure 55, 1 hour after administration. The top panel shows images of the tumor surface and center, and the bottom panel shows areas of high and low signal intensity, respectively. Areas of high signal intensity were observed around the blood vessels indicated by arrowheads. On the other hand, blood vessels were present even in areas with weak signal intensity, suggesting that the degree of conjugate leakage varies depending on the vessel. Furthermore, since more areas with high signal intensity were observed in the center of the tumor than in the surface, it is speculated that more leaky vessels are present in the center. Figures 56 and 57 demonstrate that the conjugate accumulation rate differs for each blood vessel within the tumor. Therefore, we analyzed the αCD44 fluorescence intensity for each blood vessel within the tumor. Fluorescence intensity was measured around all blood vessels in confocal microscopy images. Figure 58 shows a boxplot plotting the αCD44 fluorescence intensity for each blood vessel in each group (3 mice per group). The results showed that the tumor center showed higher values ​​1 hour and 24 hours after administration of the conjugate under labeling condition H. On the other hand, the tumor surface showed higher values ​​24 hours after administration of the conjugate under labeling condition L. These results suggest that the degree and area of ​​tumor accumulation differ depending on the labeling conditions, i.e., the hydrophobicity of the conjugate. Less hydrophobic conjugates are expected to accumulate in tumors early after administration via the leaky blood vessels that are abundant in the tumor center.On the other hand, more hydrophobic conjugates are expected to accumulate in tumors over a certain period of time after administration via the less leaky blood vessels present at the tumor surface. This suggests that the optimal irradiation timing and area for antitumor efficacy may differ depending on the conjugate labeling rate. On the other hand, simultaneous administration of multiple conjugates with controlled labeling rates may allow the conjugates to accumulate over a wider area within the tumor, potentially enhancing the antitumor effect.

[0111] (Experimental Result 9) - Evaluation of the Cytotoxicity of Compound 4-5 (without Light Irradiation) - In this study, the cytotoxicity of Compound 4-5 was evaluated. Compound 4-5 was dissolved in DMSO to prepare a stock solution of Compound 4-5 at concentrations of 250 μM and 1 mM. Cultured tumor cells (mouse colon tumor cell line: Colon26) suspended in RPM1640 medium were seeded into a 96-well plate at 5,000 cells per well and cultured at 37°C in a 5% CO2 incubator for 24 hours. Filter-sterilized Compound 4-5 was diluted to RPMI1640 medium at 1 / 100th the volume of the 250 μM and 1 mM stock solutions to prepare final dilutions of Compound 4-5 at 2.5 μM and 10 μM, respectively. Two experimental control groups were prepared: medium without Compound 4-5 or solvent, and a solution prepared by adding 1 / 100th the volume of DMSO to the medium to obtain 1% DMSO. After incubation, the culture medium was removed from the cells, and each diluted solution was added to the cells. The treated cells were cultured at 37°C in a 5% CO2 incubator for up to 7 days. Cells were subjected to a WST8 assay on days 1, 2, 3, and 7 of incubation, and absorbance at 450 nm, indicating cell viability, was measured using a plate reader. Microscopic observations were also performed on the same day using a phase-contrast microscope. The upper panel of Figure 59 shows the WST8 assay results, plotting absorbance at 450 nm, indicating cell viability, over the number of days since the start of incubation. The left panel of Figure 59 shows the graph for compound concentrations of 2.5 μM, and the right panel shows the graph for compound concentrations of 10 μM. The lower panel of the figure plots the relative absorbance of each treatment group, with the DMSO control as the denominator and the absorbance of the control group as the numerator. The plots represent the average of three replicate experiments, and error bars indicate the standard deviation between the data. This figure does not reveal any significant changes in tumor cell viability. In this figure, days 3 to 7 correspond to the death phase of tumor cells, and a decrease in viability was observed in all groups. Figures 60 and 61 are micrographs of cells taken with a phase-contrast microscope on days 1 (top) and 3 (bottom) of culture. From the low-magnification images (10x objective lens) in Figure 60, no significant changes in cell number were observed on days 1 and 3 of culture.61, no significant changes in cell morphology were observed in any of the groups on days 1 and 3 of culture. As described above, this test did not confirm any significant cytotoxicity in Compound 4 and Compound 5 themselves, and it is presumed that these compounds are capable of coexisting with cells.

[0112] (Experimental Result 10) - Evaluation of the Cytotoxicity of Compound 3-6 (with Light Irradiation) - In this study, the cytotoxicity of Compound 3-6 due to light irradiation was evaluated. Compound 3-6 was dissolved in DMSO to prepare stock solutions at concentrations of 250 μM and 1 mM. Due to the low water solubility of Compound 3, only the 250 μM stock solution was prepared. Cultured tumor cells (mouse colon tumor cell line: Colon26) suspended in RPM1640 medium were seeded into a 96-well plate at 5,000 cells per well and cultured at 37°C in a 5% CO2 incubator for 24 hours. Filter-sterilized Compound 3-6 was diluted to RPMI1640 medium at 1 / 100, 1 / 500, and 1 / 2,500 volumes to prepare final dilutions of 2.5 μM, 0.5 μM, and 0.1 μM, respectively. Furthermore, a 100-volume diluted solution of 1 mM compound 4-6 was added to the cells to prepare a final concentration of 10 μM. For experimental controls, DMSO was diluted with RPMI 1640 medium to prepare 1%, 0.2%, and 0.04% DMSO solutions according to the respective dilution amounts. After incubation, the culture medium was removed from the cells, and each solution was added to the cells. The cells were then cultured for 1 hour at 37°C in a 5% CO2 incubator. The non-washed group was treated in the diluted solution, while the washed group was cultured in the diluted solution, where the diluted solution was removed and RPMI 1640 medium was added again. To evaluate the cytotoxicity of compounds excited by light irradiation, an LED light source with a central wavelength of 730 nm, matching the maximum absorption wavelength of compound 3-6, was used at an output of 12 mW / cm. 2 , energy amount 21.6 J / cm 2The cells were irradiated under the following conditions. One hour and 24 hours after light irradiation, a WST8 assay was performed on the cells, and the absorbance at 450 nm, indicating cell viability, was measured. Microscopic observations were also performed on the same day using a phase-contrast microscope. Figure 62 shows the results of the WST8 assay, plotting the absorbance at 450 nm, indicating cell viability, on the Y-axis and the final concentration of each compound on the X-axis. The experimental control DMSO concentrations were plotted at 1% (10 μM and 2.5 μM control groups), 0.2%, and 0.04% for compound concentrations of 10 μM, 2.5 μM, 0.5 μM, and 0.1 μM, respectively. Figure 63 plots the relative absorbance of each treatment group, with the absorbance of the control group as the denominator and the absorbance of the treatment group as the numerator. The plots are the average values ​​of three replicate experiments, and the error bars indicate the standard deviation between the data. As shown in this figure, 1 hour after light exposure, a significant decrease in viability was observed with 0.5 μM compounds 3 and 6. This decrease in viability was observed in both the unwashed and washed groups. In the unwashed group, 24 hours after light exposure, a significant decrease in viability was observed with compounds 3 and 6 at 0.1 μM and with compounds 4 and 5 at 2.5 μM. In the washed group, only compounds 3 and 6 exhibited a significant decrease in viability at 0.5 μM. Figure 64 shows microscopic images of cells taken with a phase-contrast microscope (40x objective) 24 hours after light exposure. In the unwashed group, 24 hours after light exposure, cells exhibited spherical morphology and swollen cells, indicating cell death, were observed with compounds 3 and 6 at 0.1 μM and with compounds 4 and 5 at 2.5 μM. In the washed group, only compounds 3 and 6 exhibited spherical morphology and swollen cells, indicating cell death, at 0.5 μM. No significant changes in cell morphology were observed in the other groups. Furthermore, the cytotoxicity of Compound 6 at lower concentrations was evaluated. Using the same procedure as above, a 5 μM stock solution was prepared in addition to the 250 μM, and the cytotoxicity of Compound 6 was evaluated at 0.005, 0.01, 0.05, and 0.1 μM. The left side of Figure 65 shows a graph plotting the absorbance at 450 nm, which indicates cell viability, from a WST8 assay performed 1 hour and 24 hours after irradiation. The plot is the average of two replicate experiments, and the error bars indicate the standard deviation between the data.The right side shows a microscopic image taken with a phase-contrast microscope (10x objective). This figure confirms that the unwashed group exhibited significant cytotoxicity at concentrations above 0.05 μM. The washed group exhibited weaker cytotoxicity at concentrations below 0.1 μM. These results demonstrate that compounds 3-6 all exhibit cytotoxicity upon light irradiation. Compounds 3 and 6 in particular exhibited significant cytotoxicity even at lower concentrations, with compound 3 exhibiting cytotoxicity at concentrations of at least 0.1 μM and compound 6 exhibiting cytotoxicity at concentrations above 0.05 μM. Furthermore, compounds 4 and 5 showed no cytotoxicity in the washed group, suggesting that they do not possess cell membrane permeability. On the other hand, compounds 3 and 6 exhibited cytotoxicity even in the washed group, suggesting that they possess cell membrane permeability.

[0113] (Experimental Result 11) - Evaluation of the Cytotoxicity of Compound 7 (without Light Irradiation) - In this study, the cytotoxicity of Compound 7 was evaluated. Compound 7 was dissolved in DMSO to a concentration of 250 μM to prepare a stock solution. Cultured tumor cells (mouse colon tumor cell line: Colon26) suspended in RPM1640 medium were seeded into a 96-well plate at 5,000 cells per well and cultured at 37°C in a 5% CO2 incubator for 24 hours. A 1 / 100 volume of a filter-sterilized 250 μM stock solution of Compound 7 was added to RPMI1640 medium to prepare diluted solutions with a final concentration of 2.5 μM. Two experimental control groups were prepared: medium without Compound 7 or solvent, and a solution prepared by adding 1 / 100 volume of DMSO to the medium to achieve a 1% DMSO concentration. After incubation, the culture medium was removed from the cells, and each diluted solution was added to the cells. The treated cells were cultured at 37°C in a 5% CO2 incubator for up to 7 days. Cells were subjected to a WST8 assay on days 1, 2, 3, and 7 of culture, and absorbance at 450 nm, indicating cell viability, was measured using a plate reader. Microscopic observations were also performed on the same day using a phase-contrast microscope. Figure 66 shows phase-contrast images of cells on days 1 (top) and 3 (bottom). Low-magnification images (10x objective lens) in Figure 66 reveal no significant differences in cell number between the 1% DMSO-treated group and the Compound 7-treated group on day 3 of culture. High-magnification images also reveal no abnormal cell morphology. Figure 67 shows a graph of absorbance at 450 nm, indicating cell viability, plotted against days since the start of culture after a WST8 assay. The plots represent the average values ​​of three replicate experiments, and error bars indicate the standard deviation between the data. From this figure, no significant difference in survival rate was observed between the 1% DMSO-added group and the compound 7-added group. From the above, no significant cytotoxicity was confirmed in compound 7 itself, and it is presumed that it is a compound that can coexist with cells.

[0114] (Experimental Result 12) - Evaluation of the cytotoxicity of compound 7 (with light irradiation) - In this study, the cytotoxicity of compound 7 due to light irradiation was evaluated. Compound 7 was dissolved in DMSO to a concentration of 250 μM to prepare a stock solution. Cultured tumor cells (mouse colon tumor cell line: Colon26) suspended in RPM1640 medium were seeded into a 96-well plate at 5,000 cells per well and cultured at 37°C in a 5% CO2 incubator for 24 hours. Filter-sterilized compound 7 from the 250 μM stock solution was added to RPMI1640 medium at 1 / 100, 1 / 500, and 1 / 2,500 volumes to prepare diluted solutions with final concentrations of 2.5 μM, 0.5 μM, and 0.1 μM, respectively. In addition, as experimental controls, DMSO was diluted with RPMI 1640 medium to prepare 1%, 0.2%, and 0.04% DMSO diluted solutions according to the respective dilution amounts. After culturing, the culture medium was removed from the cells, and each solution was added to the cells. The cells treated as described above were cultured at 37°C for 1 hour in a 5% CO2 incubator. The group in which the next treatment was performed in the diluted solution was designated the non-washed group, while the group in which the diluted solution was removed from the cultured cells and only RPMI 1640 medium was added again was designated the washed group. To evaluate the cytotoxicity of compounds excited by light irradiation, an LED light source with a central wavelength of 660 or 730 nm was used, with an output of 12 mW / cm. 2 , energy amount 21.6 J / cm 2Cells were irradiated under the following conditions. Cells were subjected to a WST8 assay 1 and 24 hours after light irradiation, and absorbance at 450 nm was measured to indicate cell viability. On the same day, microscopic observations were performed using a phase-contrast microscope. Figure 68 shows microscopic images of cells irradiated with 660 nm light taken with a phase-contrast microscope (40x objective) 1 hour (left) and 24 hours (right) after irradiation. This figure shows that cells exhibited spherical morphology and swollen cells, indicating cell death, in the 2.5 μM unwashed and washed groups. Figure 69 shows microscopic images of cells irradiated with 730 nm light taken with a phase-contrast microscope (40x objective) 1 hour (left) and 24 hours (right). This figure shows that cells exhibited spherical morphology and swollen cells, indicating cell death, in the 0.5 and 2.5 μM unwashed groups 1 and 24 hours after irradiation. In addition, cells with spherical morphology and swollen cells, indicating cell death, were observed in the 2.5 μM washed group 1 and 24 hours after irradiation. Note that no significant changes in cell morphology were observed in the control and 0.1 μM groups in Figures 68 and 69. Figures 70 and 71 show graphs of the WST8 assay, plotting the absorbance at 450 nm (indicating cell viability) on the Y-axis and the final concentration of each compound on the X-axis. The control DMSO concentrations were plotted at 1%, 0.2%, and 0.04% for compound concentrations of 2.5 μM, 0.5 μM, and 0.1 μM, respectively. The plots represent the average values ​​of four replicate experiments, and the error bars indicate the standard deviation between the data. Figure 70, which shows cell viability following 660 nm light irradiation, reveals a significant decrease in cell viability only in the 2.5 μM unwashed and washed groups 24 hours after irradiation. Furthermore, a slight decrease in cell viability was observed in the 2.5 μM unwashed group 1 hour after irradiation. Figure 71, which shows cell viability after 730 nm light irradiation, shows a significant decrease in cell viability only in the 2.5 μM unwashed and washed groups 24 hours after irradiation. Furthermore, a slight decrease in cell viability was observed in the 2.5 μM unwashed and washed groups 1 hour after irradiation, and in the 0.5 μM unwashed and washed groups 24 hours after irradiation.The results of phase-contrast microscopy and the WST8 assay generally agreed, confirming that compound 7 exhibited significant cytotoxicity upon irradiation with light at a wavelength corresponding to its maximum absorption wavelength. Furthermore, the WST8 assay results suggest that its effective concentration is approximately 2.5 μM. Furthermore, significant cytotoxicity was also observed upon irradiation with light at a wavelength (70 nm) outside the maximum absorption wavelength. Furthermore, the cytotoxicity observed in the washed group suggests that compound 7 possesses cell permeability.

[0115] The disclosure of Japanese Patent Application No. 2024-099175, filed on June 19, 2024, is incorporated herein by reference in its entirety. All documents, patent applications, and technical standards mentioned herein are incorporated herein by reference to the same extent as if each individual document, patent application, and technical standard was specifically and individually indicated to be incorporated by reference.

Claims

1. A tumor cell killing agent represented by the following general formula (I): (In general formula (I), X １ ~X ８ each independently represents a hydrogen atom or a substituent (excluding a halogen atom). １ and X ２ , X ３ and X ４ , X ５ and X ６ and X ７ and X ８ may each independently be linked to each other via a substituent to form a benzene ring. Y represents an oxygen atom or a sulfur atom. R １ ~R ８ each independently represents an organic group, provided that when Y is an oxygen atom, R １ ~R ８ Not all of the groups are phenyl groups.) 2. In general formula (I), X １ ~X ８ 2. The tumor cell killing agent according to claim 1, wherein is a hydrogen atom and Y is an oxygen atom.

3. In general formula (I), R １ ~R ８ The tumor cell killing agent according to claim 2, wherein at least one of the organic groups represented by the formula (I) has a hydroxy group.

4. In general formula (I), R １ ~R ８ The tumor cell killing agent according to claim 2, wherein at least one of the organic groups represented by the formula (I) has a carboxy group or an alkali metal salt thereof.

5. In general formula (I), R １ ~R ８ The tumor cell killing agent according to claim 2, wherein at least one of the organic groups represented by the formula (I) has a maleimide group.

6. In general formula (I), R １ ~R ８ The organic group represented by -(R ９ O) ｎ -R １０ (wherein R ９ represents an alkylene group, and R １０ The tumor cell killing agent according to claim 2, wherein: represents an alkyl group; and n represents an integer of 1 or more.

7. In general formula (I), R １ ~R ８ 3. The tumor cell killing agent according to claim 2, wherein the organic group represented by the formula: is an alkyl group having a total of 1 to 18 carbon atoms.

8. In general formula (I), X １ ~X ８ The tumor cell killing agent according to claim 1, wherein each of the groups independently represents an alkoxy group or an aryloxy group, and Y represents an oxygen atom.

9. In general formula (I), R １ ~R ８ The tumor cell killing agent according to claim 8, wherein at least one of the organic groups represented by the formula (I) has a hydroxy group.

10. In general formula (I), R １ ~R ８ 9. The tumor cell killing agent according to claim 8, wherein at least one of the organic groups represented by the formula (I) has a carboxy group or an alkali metal salt thereof.

11. In general formula (I), R １ ~R ８ The tumor cell killing agent according to claim 8, wherein at least one of the organic groups represented by the formula (I) has a maleimide group.

12. In general formula (I), R １ ~R ８ The organic group represented by -(R ９ O) ｎ -R １０ (wherein R ９ represents an alkylene group, and R １０ The tumor cell killing agent according to claim 8, wherein: represents an alkyl group; and n represents an integer of 1 or more.

13. In general formula (I), X １ ~X ８ The tumor cell killing agent according to claim 1, wherein each of the groups independently represents a thioalkoxy group or a thioaryloxy group, and Y represents an oxygen atom.

14. In general formula (I), X １ and X ２ , X ３ and X ４ , X ５ and X ６ and X ７ and X ８ The tumor cell killing agent according to claim 1, wherein each of the groups is linked to each other via a substituent to form a benzene ring, and Y is an oxygen atom.

15. In general formula (I), R １ ~R ８ The tumor cell killing agent according to claim 14, wherein at least one of the organic groups represented by the formula (I) has a hydroxy group.

16. In general formula (I), R １ ~R ８ The tumor cell killing agent according to claim 14, wherein at least one of the organic groups represented by the formula (I) has a carboxy group or an alkali metal salt thereof.

17. In general formula (I), R １ ~R ８ The tumor cell killing agent according to claim 14, wherein at least one of the organic groups represented by the formula (I) has a maleimide group.

18. In general formula (I), R １ ~R ８ The organic group represented by -(R ９ O) ｎ -R １０ (wherein R ９ represents an alkylene group, and R １０ The tumor cell killing agent according to claim 14, wherein: represents an alkyl group; and n represents an integer of 1 or more.

19. In general formula (I), X １ ~X ８ 2. The tumor cell killing agent according to claim 1, wherein is a hydrogen atom and Y is a sulfur atom.

20. The solubility in water at 25°C is 0.1 g / 100 g-H ２ The tumor cell killing agent according to claim 1, wherein the cytotoxicity is O or more.

21. A tumor cell killing agent-antibody conjugate in which the tumor cell killing agent of claim 4 is conjugated to the antibody via an amide bond formed between a carboxy group or an alkali metal salt thereof possessed by the tumor cell killing agent and an amino group contained in an antibody having an amino group.

22. A tumor cell killing agent-antibody conjugate in which the tumor cell killing agent of claim 5 is conjugated to the antibody via a thioether bond formed between a maleimide group contained in the tumor cell killing agent and a thiol group contained in an antibody having a thiol group.

23. The tumor cell killing agent-antibody conjugate of claim 21 or claim 22, wherein the antibody is anti-CD44 rat IgG.

Images