Phthalocyanine dye, method for producing same, and drug containing phthalocyanine dye

Abstract

Provided are a phthalocyanine dye represented by formula (1) and a method for producing the same. (In the formula, M, L, Q, R2, R3, R7, R8, R4, R5, R6, R9, R10, R11, R12, R13, R14, R15, R16, R17, R18, R19, R20, R21, R22, R23, X2, and X3 are as defined in the claims.)

Description

Phthalocyanine dye, its manufacturing method, and pharmaceutical agent containing phthalocyanine dye

[0001] The present invention relates to a phthalocyanine dye, a method for producing the same, and a pharmaceutical agent containing the phthalocyanine dye.

[0002] Photoimmunotherapy, a cutting-edge cancer treatment method that has been attracting attention in recent years, uses a photoimmunotherapy drug that is a conjugate of a water-soluble phthalocyanine silicon complex and an antibody.

[0003] Patent Document 1 discloses a water-soluble silicon phthalocyanine, and indicates that biologically relevant substances such as antibodies can be labeled with the phthalocyanine dye of Patent Document 1 and used in Western blots and the like.

[0004] Patent Document 2 discloses a method for inducing cytotoxicity in a subject suffering from a disease or condition, the method comprising: (a) administering to the subject a therapeutically effective agent comprising a phthalocyanine dye, such as IRDye® 700DX (hereinafter also simply referred to as “IR700”), conjugated to a probe that specifically binds to cells of the subject; and (b) irradiating the cells with appropriate excitation light in an amount effective to induce cell death.

[0005]

[0006] It has been shown that target cells can be destroyed by using antibodies labeled with phthalocyanine silicon complexes such as IR700 as photosensitizers.

[0007] Non-Patent Document 1 discloses photoimmunotherapy for an EGFR-expressing cancer cell model using a probe combining IR700 and an anti-EGFR antibody.

[0008] Non-Patent Document 2 discloses that, in order to avoid the complicated synthesis of IR700, which has an asymmetric phthalocyanine skeleton, a novel silicon phthalocyanine-antibody conjugate was synthesized using an axially substituted phthalocyanine skeleton, in which the antibody was bound to the antibody via an axial linker, and that this was applied as a photosensitizer to kill HER2-overexpressing KPL-4 cells.

[0009] WO 2004 / 038378 A2WO 2015 / 187677 A1

[0010] Molecular Oncology 2014, 3, 620-632, DOI:10.1016 / j.molonc.2014.01.006Synlett 2021 32, 1098-1103, DOI: 10.1055 / a-1503-6425

[0011] The silicon phthalocyanines in Patent Document 1, Patent Document 2, Non-Patent Document 1, and Non-Patent Document 2 are all compounds in which a linker is bonded to the phthalocyanine skeleton at the ortho position via an ether group. Claim 12 of Patent Document 1 describes a compound in which a linker is bonded to the phthalocyanine skeleton at the meta position, but according to the detailed explanation in the specification, such a compound has not been synthesized.

[0012] Furthermore, conventional synthetic routes for water-soluble silicon phthalocyanines, such as IR700, a water-soluble silicon phthalocyanine used in photoimmunotherapy drugs, are technically challenging and difficult to synthesize. If IR700 or its derivatives can be synthesized more simply and efficiently than conventional methods, it is expected that this will lead to the development of various photoimmunotherapy drugs that are effective against cancers that have not been treated to date.

[0013] The problem to be solved by the present invention is to provide a novel phthalocyanine dye, a method for producing the novel phthalocyanine dye, and a pharmaceutical agent containing the phthalocyanine dye.

[0014] The present invention includes the following embodiments: Item 1. A phthalocyanine dye represented by formula (I).

[0015]

[0016] wherein M is selected from Si, Ge, Sn, or Al; L is a linker comprising an optionally substituted triazolyldiyl group or a fused ring of an optionally substituted diazine and a cycloalkane; Q is a reactive group for attachment of the dye to a targeting molecule; and R 2 , R 3 , R7 , and R 8 are each independently selected from optionally substituted alkyl and optionally substituted aryl; R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 are each independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkanoyl, optionally substituted alkoxycarbonyl, optionally substituted alkylcarbamoyl, and a chelating ligand, wherein R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 at least one of R contains a water-soluble group; 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 and R 23 are each independently selected from hydrogen, halogen, optionally substituted alkylthio, and optionally substituted alkylamino, and X 1 and X 2 each independently represents a C-C group optionally interrupted by a heteroatom; 10 Item 2. The phthalocyanine dye according to Item 1, wherein L is a group represented by the following formula (II):

[0017]

[0018] wherein Y is selected from a direct bond, oxygen, sulfur, or optionally substituted nitrogen; and X 3 and X 4 each independently represents a C-C group optionally interrupted by a heteroatom; 10(Item 3) Q is selected from alkylene, polyethylene glycol (PEG), phenylene, ester, amide, carbamate, or a combination of two or more thereof, and Z is an optionally substituted triazolyldiyl group or a fused ring of an optionally substituted diazine and a cycloalkane. Item 3. The phthalocyanine dye according to Item 1, wherein Q is selected from an amine-reactive chemical group, a sulfhydryl-reactive chemical group, an activated ester, an acyl halide, an alkyl halide, an acid anhydride group, carboxy, carboxylate, a carboxylate, a carbodiimide, a carbonate, a carbamate, a haloacetamide, an isocyanate, an isothiocyanate, a maleimide, an N-hydroxysuccinimide ester, a phosphoramidite, a platinum complex, a sulfonate ester, and a thiocyanate. Item 4. A pharmaceutical agent comprising the phthalocyanine dye according to any one of Items 1 to 3, conjugated to a targeting molecule that binds to an antigen, a protein, or a cell surface target molecule. Item 5. The pharmaceutical agent according to Item 4, wherein the targeting molecule is a protein, a peptide, or a small molecule. Item 6. Item 4. The agent according to Item 4, wherein the targeting molecule is an antibody or an antibody fragment. Item 7. The agent according to Item 4, for inducing cytotoxicity by apoptosis in target cells. Item 8. The agent according to Item 7, wherein the induction of cytotoxicity is brought about by administering the agent to the tumor cells and irradiating the tumor cells with light after administering the agent. Item 9. The agent according to Item 4, for treating a tumor in a target. Item 10. A method for producing a phthalocyanine dye represented by formula (IV), comprising reacting a compound represented by formula (III) with reagent (B) to obtain a compound represented by formula (IV).

[0019]

[0020] (In formulas (III) and (IV), M is selected from Si, Ge, Sn, or Al; R 2 , R 3 , R 7 , and R 8 are each independently selected from optionally substituted alkyl and optionally substituted aryl; R 4 , R 5 , R 6 , R9 , R 10 , and R 11 are each independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkanoyl, optionally substituted alkoxycarbonyl, optionally substituted alkylcarbamoyl, and a chelating ligand, wherein R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 at least one of R contains a water-soluble group; 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 and R 23 are each independently selected from hydrogen, halogen, optionally substituted alkylthio, and optionally substituted alkylamino; Y is selected from a direct bond, oxygen, sulfur, or optionally substituted nitrogen; X 1 and X 2 each independently represents a C-C group optionally interrupted by a heteroatom; 10 is alkylene, and X 3 and X 4 each independently represents a C-C group optionally interrupted by a heteroatom; 10 selected from alkylene, polyethylene glycol (PEG), phenylene, ester, amide, carbamate, or a combination of two or more thereof, Z is an optionally substituted triazolyldiyl group or a fused ring of an optionally substituted diazine and a cycloalkane, P 1is selected from an amine-reactive chemical group, a sulfhydryl-reactive chemical group, an activated ester, an acyl halide, an alkyl halide, an acid anhydride group, carboxy, carboxylate, a carboxylate, a carbodiimide, a carbonate, a carbamate, a haloacetamide, an isocyanate, an isothiocyanate, a maleimide, an N-hydroxysuccinimide ester, a phosphoramidite, a platinum complex, a sulfonate ester, and a thiocyanate, A has a first functional group, and reagent (B) has a second functional group that reacts with the first functional group of A by a click reaction.) Item 11. A method for producing a phthalocyanine dye represented by formula (VII), comprising reacting a compound represented by formula (VI) with an azide (X) to obtain a compound represented by formula (VII).

[0021]

[0022] In formulas (VI), (VII), and (X), M is selected from Si, Ge, Sn, or Al; R 2 , R 3 , R 7 , and R 8 are each independently selected from optionally substituted alkyl and optionally substituted aryl; R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 are each independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkanoyl, optionally substituted alkoxycarbonyl, optionally substituted alkylcarbamoyl, and a chelating ligand, wherein R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 at least one of R contains a water-soluble group; 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20, R 21 , R 22 and R 23 are each independently selected from hydrogen, halogen, optionally substituted alkylthio, and optionally substituted alkylamino; Y is selected from a direct bond, oxygen, sulfur, or optionally substituted nitrogen; X 1 and X 2 each independently represents a C-C group optionally interrupted by a heteroatom; 10 is alkylene, and X 4 each independently represents a C-C group optionally interrupted by a heteroatom; 10 Z is an optionally substituted triazolyldiyl group selected from alkylene, polyethylene glycol (PEG), phenylene, ester, amide, carbamate, or a combination of two or more thereof; P 1 is selected from among an amine-reactive chemical group, a sulfhydryl-reactive chemical group, an activated ester, an acyl halide, an alkyl halide, an acid anhydride group, carboxy, carboxylate, a carboxylate salt, a carbodiimide, a carbonate, a carbamate, a haloacetamide, an isocyanate, an isothiocyanate, a maleimide, an N-hydroxysuccinimide ester, a phosphoramidite, a platinum complex, a sulfonate ester, and a thiocyanate.) Item 12. A method for producing a phthalocyanine dye represented by formula (I), comprising producing a compound represented by formula (IV) by the method described in item 10, and adding P of formula (IV) to the compound represented by formula (IV) thus produced. 1 or preparing a compound represented by formula (VII) by the method according to claim 11, and reacting the compound represented by formula (VII) with a compound having a reactive functional group to obtain a compound represented by formula (I): 1 and reacting the group with a compound having a reactive functional group to obtain a compound represented by formula (I):

[0023]

[0024] (In formula (I), M is selected from Si, Ge, Sn, or Al, L is a group represented by the following formula (II), -X 4 -ZX 3 -Y- Formula (II) (wherein Y is selected from a direct bond, oxygen, sulfur, or optionally substituted nitrogen; X 3 and X 4 each independently represents a C-C group optionally interrupted by a heteroatom; 10 R is a group selected from alkylene, polyethylene glycol (PEG), phenylene, ester, amide, carbamate, or a combination of two or more thereof; Z is an optionally substituted triazolyldiyl group or an optionally substituted fused ring of diazine and cycloalkane; Q is a reactive group for attachment of the dye to a targeting molecule; 2 , R 3 , R 7 , and R 8 are each independently selected from optionally substituted alkyl and optionally substituted aryl; R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 are each independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkanoyl, optionally substituted alkoxycarbonyl, optionally substituted alkylcarbamoyl, and a chelating ligand, wherein R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 at least one of R contains a water-soluble group; 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 and R 23are each independently selected from hydrogen, halogen, optionally substituted alkylthio, and optionally substituted alkylamino, and X 1 and X 2 each independently represents a C-C group optionally interrupted by a heteroatom; 10 Item 13. Any of the following compounds (iii) to (vi):

[0025]

[0026] (wherein M is selected from Si, Ge, Sn, or Al; R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 and R 23 are each independently selected from hydrogen, halogen, optionally substituted alkylthio, and optionally substituted alkylamino; Y is selected from a direct bond, oxygen, sulfur, or optionally substituted nitrogen; and X 3 each independently represents a C-C group optionally interrupted by a heteroatom; 10 alkylene.)

[0027]

[0028] (In formula (iv), M is selected from Si, Ge, Sn, or Al; R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 and R 23 are each independently selected from hydrogen, halogen, optionally substituted alkylthio, and optionally substituted alkylamino; R 24 and R25 are each independently C1-C 10 an alkyl group, and Y is selected from a direct bond, oxygen, sulfur, or an optionally substituted nitrogen; X 3 each independently represents a C-C group optionally interrupted by a heteroatom; 10 alkylene.)

[0029]

[0030] (wherein M is selected from Si, Ge, Sn, or Al; R 2 , R 3 , R 7 , and R 8 are each independently selected from optionally substituted alkyl and optionally substituted aryl; R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 and R 23 are each independently selected from hydrogen, halogen, optionally substituted alkylthio, and optionally substituted alkylamino, and Y is selected from a direct bond, oxygen, sulfur, or optionally substituted nitrogen; X 1 and X 2 each independently represents a C-C group optionally interrupted by a heteroatom; 10 is alkylene, and X 3 each independently represents a C-C group optionally interrupted by a heteroatom; 10 alkylene.)

[0031]

[0032] (wherein M is selected from Si, Ge, Sn, or Al; R 2 , R 3 , R 7 , and R 8are each independently selected from optionally substituted alkyl and optionally substituted aryl; R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 are each independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkanoyl, optionally substituted alkoxycarbonyl, optionally substituted alkylcarbamoyl, and a chelating ligand, wherein R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 at least one of R contains a water-soluble group; 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 and R 23 are each independently selected from hydrogen, halogen, optionally substituted alkylthio, and optionally substituted alkylamino, and Y is selected from a direct bond, oxygen, sulfur, or optionally substituted nitrogen; X 1 and X 2 each independently represents a C-C group optionally interrupted by a heteroatom; 10 is alkylene, and X 3 each independently represents a C-C group optionally interrupted by a heteroatom; 10 alkylene.)

[0033] The method for producing a phthalocyanine dye of the present invention uses a synthetic intermediate different from conventional methods, and therefore enables the synthesis of a group of compounds having a structure similar to that of IR700 and photosensitizing and / or photoimmunoactivity more efficiently than conventional methods. The produced phthalocyanine dye can be used to induce cytotoxicity through apoptosis in tumor cells, treat target tumors, etc.

[0034] (A) Attenuation of absorbance at 681 nm for compound 7 upon irradiation with light. (B) Attenuation of absorbance at 678 nm for compound 14 upon irradiation with light. (C) Attenuation of fluorescence intensity at 695 nm for compound 7 upon irradiation with light. (D) Attenuation of fluorescence intensity at 687 nm for compound 14 upon irradiation with light. Cytotoxicity test of DiFi-BRAF cells by compound 8 and irradiation with light. Antibody concentration-dependent cytotoxicity test of DiFi-BRAF cells by compound 8-cet or compound 15-cet and irradiation with light. n=4 per group. Energy-dependent cytotoxicity test of DiFi-BRAF cells by compound 8-cet or compound 15-cet and irradiation with light. n=4 per group. HPLC analysis of the degradation of compound 7 upon near-infrared light irradiation. Morphological changes of DiFi(BRAF) cells treated with compound 8-cetuximab upon near-infrared light irradiation. Arrows indicate bleb formation. Viability assessment of HTLV-1-infected / uninfected cells using compound 8-bas and near-infrared light. Light-induced cytotoxicity of compound 8-bas in a mixed system of HTLV-1-infected / uninfected cells was assessed by FACS. (Left) Without near-infrared light irradiation, (Right) With near-infrared light irradiation (32 J / cm). 2 ) FACS evaluation of light-induced cytotoxicity of the compound 8-basiliximab against blood-derived cells from ATL patients. (Left) Without near-infrared light irradiation, (Right) With near-infrared light irradiation (64 J / cm 2 )

[0035] As used herein, the singular forms (a, an, the) include both the singular and the plural unless otherwise expressly stated herein or clearly contradicted by context.

[0036] In this specification, the term "comprise" is a concept that encompasses "consist essentially of" and "consist only of."

[0037] Unless otherwise specified herein, examples of "substituents" include halogen atoms, hydroxyl groups, cyano groups, nitro groups, alkyl groups, hydroxyalkyl groups, halogenoalkyl groups, cycloalkyl groups, hydroxycycloalkyl groups, cycloalkyl-alkyl groups, aralkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, halogenoalkoxy groups, cycloalkoxy groups, cycloalkyl-alkoxy groups, alkyl sulfoxides, aryl sulfoxides, sulfo groups, alkylthio groups, cycloalkyl-alkylthio groups, amino groups, mono- or dialkylamino groups, dialkylamino-alkyl groups, cycloalkylamino groups, cycloalkyl-alkylamino groups, acyl groups, acyloxy groups, oxo groups, carboxyl groups, alkoxycarbonyl groups, aralkyloxycarbonyl groups, carbamoyl groups, saturated or unsaturated heterocyclic groups, unsaturated hydrocarbon ring groups such as aromatic hydrocarbon groups, saturated heterocyclic oxy groups, etc. When substituents are present, the number thereof is typically one, two, or three.

[0038] As used herein, C1-C 10 indicates a group having 1 to 10 carbon atoms.

[0039] As used herein, C1-C optionally interrupted by heteroatoms 10 Alkylene does not include polyethylene glycol (PEG).

[0040] As used herein, a strained alkene refers to a molecule containing an alkene moiety that has one or more double bonds that are bent due to ring strain and that is capable of reacting with a tetrazine.

[0041] As used herein, strained alkynes refer to molecules containing an alkyne moiety that has one or more triple bonds bent due to ring strain and that are capable of reacting with tetrazines.

[0042] As used herein, the term "cancer" refers to a physiological condition in mammals that is generally characterized by unregulated cell proliferation. Non-limiting examples of cancer include carcinoma, lymphoma, blastoma, sarcoma, and leukemia or malignant lymphoma. More specific examples of cancer include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer including small cell lung cancer, non-small cell lung cancer (NSCLC), lung adenocarcinoma and squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastric cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal region cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatocellular carcinoma, anal carcinoma, penile carcinoma, and head and neck cancer.

[0043] As used herein, the term "tumor" includes solid tumors, which refer to an abnormal mass of cells that does not contain a cyst, whether benign or malignant. Non-limiting examples of solid tumors include glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0044] As used herein, the term "subject" or "patient" refers to a mammal, such as a human, mouse, rat, guinea pig, hamster, rabbit, monkey, dog, cat, cow, pig, horse, sheep, etc.

[0045] Prior to completing the present invention, the present inventors attempted to produce the following asymmetric silicon phthalocyanine (SiPC) based on the descriptions in prior art such as WO 2004 / 038378 A2, but were unable to synthesize it.

[0046]

[0047] Therefore, the inventors have succeeded in synthesizing an asymmetric phthalocyanine skeleton more efficiently than conventional methods by changing the position of the linker used to bind the targeting molecule to the phthalocyanine skeleton from the ortho position to the meta position.

[0048] Although the present invention does not wish to be bound by a particular theory, it is believed that one of the reasons why silicon phthalocyanine was synthesized by changing the linker position from the ortho position to the meta position is that the steric hindrance in the synthesis of the skeleton was alleviated.

[0049] Furthermore, by using click chemistry, the linker length could be easily changed, and the silicon phthalocyanine of the present invention can be easily derivatized via linker extension.

[0050] While IR700 requires 10 steps to synthesize, the synthesis method of the present invention utilizes click chemistry, shortening the number of steps from the starting material to the synthesis of silicon phthalocyanine to as few as eight.

[0051] According to one aspect of the present invention, there is provided a phthalocyanine dye represented by formula (I):

[0052]

[0053] wherein M is selected from Si, Ge, Sn, or Al; L is a linker comprising an optionally substituted triazolyldiyl group or a fused ring of an optionally substituted diazine and a cycloalkane; Q is a reactive group for attachment of the dye to a targeting molecule; and R 2 , R 3 , R 7 , and R 8 are each independently selected from optionally substituted alkyl and optionally substituted aryl; R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 are each independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkanoyl, optionally substituted alkoxycarbonyl, optionally substituted alkylcarbamoyl, and a chelating ligand, wherein R 4 , R 5 , R 6 , R 9 , R 10 , and R 11at least one of R contains a water-soluble group; 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 and R 23 are each independently selected from hydrogen, halogen, optionally substituted alkylthio, and optionally substituted alkoxy; and X 1 and X 2 each independently represents a C-C group optionally interrupted by a heteroatom; 10 It is alkylene.

[0054] R 2 , R 3 , R 7 , and R 8 When has a substituent, the substituent is not particularly limited, and may be any of the substituents defined in this specification.

[0055] When L is a linker containing a triazolyldiyl group as a substituent, the substituent is not particularly limited, and may be any of the substituents defined herein.

[0056] When L is a linker containing a fused ring of an optionally substituted diazine and a cycloalkane, the substituent is not particularly limited, and may be any of the substituents defined in this specification.

[0057] R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 When has a substituent, the substituent is not particularly limited, and may be any of the substituents defined in this specification.

[0058] R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R20 , R 21 , R 22 and R 23 When has a substituent, the substituent is not particularly limited, and may be any of the substituents defined in this specification.

[0059] X 3 and X 4 each of which is optionally interrupted by a heteroatom; 10 When alkylene, the heteroatom is, for example, N, O, or S.

[0060] Phthalocyanines are a group of photosensitizer compounds that contain a phthalocyanine ring system. Phthalocyanines are azaporphyrins (i.e., C ) containing four benzoindole groups connected by nitrogen bridges in a 16-membered ring of alternating carbon and nitrogen atoms. 32 H 16 N8), which forms stable chelates with metal and nonmetal cations.

[0061] In some embodiments, the phthalocyanine dyes of Formula (I) have strong red or near-infrared absorption, with an absorption peak between about 600 and 810 nm, which in some cases allows for deep tissue penetration of light. The phthalocyanine dyes of Formula (I) are photostable.

[0062] In some embodiments, the phthalocyanine dyes represented by Formula (I) are water-soluble and contain a light-emitting fluorophore moiety having at least one aqueous-solubilizing moiety.

[0063] In some embodiments, L is a covalent linkage. In some embodiments, the covalent linkage is a straight-chain or branched, cyclic or heterocyclic, saturated or unsaturated covalent linkage having 1 to 60 atoms, e.g., 1 to 45 atoms or 1 to 25 atoms. Generally, the linkage contains an ether, thioether, amine, ester, carbamate, urea, thiourea, oxy, or amide bond; or a single, double, triple, or aromatic carbon-carbon bond; or a phosphorus-oxygen, phosphorus-sulfur, nitrogen-nitrogen, nitrogen-oxygen, or nitrogen-platinum bond; or any combination of aromatic or heteroaromatic bonds.

[0064] In some embodiments, L is a group represented by formula (II):

[0065]

[0066] wherein Y is selected from a direct bond, oxygen, sulfur, or optionally substituted nitrogen; X 3 and X 4 each independently represents a C-C group optionally interrupted by a heteroatom; 10 is selected from alkylene, polyethylene glycol (PEG), phenylene, ester, amide, carbamate, or a combination of two or more thereof, and Z is an optionally substituted triazolyldiyl group.

[0067] In some embodiments, in Formula (II), Y is oxygen.

[0068] In some embodiments, in Formula (II), Y is sulfur.

[0069] In some embodiments, L comprises a linker comprising a fused ring of an optionally substituted diazine and cycloalkane, the fused ring of the diazine and cycloalkane being represented by formula (22).

[0070] In formula (22), ring A is a C3-C 10 It is cycloalkyl.

[0071] The fused ring represented by formula (22) can be formed, for example, by any of the following reactions (i) to (iii): (i) reaction of an azide with an alkyne, (ii) reaction of an azide with a strained alkyne, or (ii) reaction of a tetrazinyl with a strained alkene or strained alkyne.

[0072] In some embodiments, Q contains a reactive group for optional attachment to a targeting molecule. For example, the reactive group is an electrophile or a nucleophile that can form a covalent bond through exposure to the functional group of the corresponding targeting molecule, which is a nucleophile or an electrophile, respectively. Alternatively, the reactive group is a photoactivatable group and only becomes chemically reactive after being irradiated with light of an appropriate wavelength.

[0073] In some embodiments, Q is selected from amine-reactive chemical groups, sulfhydryl-reactive chemical groups, activated esters, acyl halides, alkyl halides, acid anhydride groups, carboxy, carboxylate, carbodiimide, carbonate, carbamate, haloacetamide, isocyanate, isothiocyanate, maleimide, N-hydroxysuccinimide (NHS) ester, phosphoramidite, platinum complex, sulfonate ester, and thiocyanate. In some embodiments, Q is reactive with carboxyl groups, amines, or thiol groups on targeting molecules. In some embodiments, Q is a sulfhydryl-reactive chemical group, such as maleimide, haloacetyl, and pyridyl disulfide. In some embodiments, Q is an amine-reactive chemical group, such as NHS ester.

[0074] In some embodiments, R 2 , R 3 , R 7 , and R 8 are each optionally substituted alkyl, for example, optionally substituted methyl, ethyl, or isopropyl groups.

[0075] In some embodiments, R 4 , R 5 , R 6 , R 9 , R 10 , and R11 At least one of R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 The alkyl portion of R is substituted with a water-soluble substituent. As used herein, a "water-soluble group" refers to a group containing one or more polar and / or ionic substituents that improve the solubility of the overall molecule in aqueous media. In some cases, R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 At least two of the groups contain water-soluble groups. In other embodiments, three or more contain water-soluble groups. Water-soluble groups include, but are not limited to, carboxylate (-CO2 - ) group, sulfonate (-SO3 - ) group, sulfonyl (-SO2 - ) group, sulfate (-SO4 -2 ) group, hydroxyl (-OH) group, phosphate (-OPO3 -2 ) group, phosphonate (-PO3 -2 ) group, an amine (-NH2) group and an optionally substituted quaternary nitrogen, each with an optional counterion.

[0076] Suitable counterions include, but are not limited to, sodium, potassium, calcium, ammonium, organic amino salts, or magnesium salts, or similar salts. Preferably, the counterion is a biologically acceptable counterion.

[0077] In some embodiments, R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 The nitrogen atom to which is attached can be trivalent or tetravalent.

[0078] In some embodiments, R 12 , R 13 , R 14 , R 15, R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 , and R 23 are each hydrogen.

[0079] In some embodiments, X 1 and X 2 are each independently a C1-C bond optionally interrupted by an atom. 10 In some embodiments, X is selected from alkylene. 1 and / or X 2 The nitrogen attached to may optionally be quaternized.

[0080] In some embodiments, the phthalocyanine dye represented by formula (I) is a phthalocyanine dye represented by formula (Ia):

[0081]

[0082] In some embodiments, the phthalocyanine dye represented by Formula (I) has a maximum light absorption in the near infrared region, ie, between 600 nm and 850 nm, e.g., between 680 nm and 850 nm, e.g., at approximately 690 nm ± 50 nm.

[0083] As used herein, "protein" refers to a polymeric compound in which 50 or more amino acids are linked together. "Peptide" refers to a compound in which two or more but less than 50 amino acids are linked together. "Small molecule" refers to a compound with a molecular weight of 900 or less, preferably 500 or less, that does not contain repeating units of the same monomer within the molecule, and does not include polymers formed by the polymerization of two or more amino acids or polymers formed by the polymerization of two or more sugars. Antigens can be proteins or peptides, but "proteins" and "peptides" as targeting molecules do not include antigens.

[0084] In some embodiments, a pharmaceutical agent is provided that contains a phthalocyanine dye of formula (I) attached or conjugated to a targeting molecule. Any targeting moiety, such as an antigen, ligand, protein, peptide, carbohydrate, nucleic acid, or small molecule, that specifically binds to a target cell can be incorporated into the cell or molecule.

[0085] A drug in which a water-soluble phthalocyanine silicon complex, a phthalocyanine dye represented by formula (I), is conjugated to a targeting molecule is useful as a new photoimmunotherapeutic drug.

[0086] In some embodiments, the targeting molecule is an antigen, a protein, or a targeting molecule that binds to a cell surface target molecule.

[0087] In some embodiments, the targeting molecule directly or indirectly binds to an antigen or protein. For example, the targeting molecule is a second binding molecule that binds to a first binding molecule, and the first binding molecule is capable of binding to the antigen or protein. In some embodiments, the targeting molecule is a second antibody.

[0088] In some embodiments, the targeting molecule binds to a cell surface target molecule on the surface of a cell or pathogen, such as a stem cell, a proliferative cell, a cancer cell, a hyperplastic cell, a tumor cell, an inflammatory cell, a neuronal cell, a pathogen, or a cell infected with a pathogen. In some embodiments, the pathogen is selected from among viruses, bacteria, fungi, biofilms, and other prokaryotic systems. In some embodiments, the cell is a cancer cell, a tumor cell, an inflammatory cell, or a neuronal cell.

[0089] In some embodiments, the cell surface target molecule comprises an antigen, a polypeptide, a lipid, or a carbohydrate, or a combination thereof.

[0090] In some embodiments, the cell surface target molecule is HER1 / EGFR, HER2 / ERBB2, CD20, CD25 (IL-2Rα receptor), CD33, CD52, CD133, CD206, CEA, CEACAM1, CEACAM3, CEACAM5, CEACAM6, cancer antigen 125 (CA 125), alpha-fetoprotein (AFP), Lewis Y, TAG72, Caprin-1, mesothelin, PDGF receptor, PD-1, PD-L1, CTLA-4, IL-2 receptor, vascular endothelial growth factor (VEGF), CD30, EpCAM, EphA2, glypican-3, gpA33, mucin, CAIX, PSMA, folate binding protein, gangliosides (e.g., GD2, GD3, GM1 and GM2), VEGF receptor (VEGFR), VEGFR2, VEGF-A, integrin α Vβ 3, integrin α 5β 1, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, tenascin, AFP, BCR complex, CD3, CD18, CD44, CTLA-4, gp72, HLA-DR10β, HLA-DR antigen, IgE, MUC-1, nuC242, PEM antigen, metalloproteinase, ephrin receptor, ephrin ligand, HGF receptor, CXCR4, CXCR4, bombesin receptor, SK-1 antigen, Bcr-abl, RET, MET, TRKB, TIE2, ALK, ROS, EML4-ALK, ROS1, BRAFV600E, SRC, c-KIT, PDGFR, mTOR, TSC1, TSC2, BTK, KIT, BRCA, CDK 4 / 6, JAK1, JAK2, BRAF, FLT-3, MEK1, MEK2, and SMO. In some embodiments, the cell surface target molecule is selected from the group consisting of HER1 / EGFR, HER2, PD-L1, CD25, EpCAM, EphA2, CD206, CD20, CD44, CD133, mesothelin, glypican-3, and carcinoembryonic antigen (CEA).

[0091] In some embodiments, the targeting molecule is or comprises a protein, a glycoprotein, an antibody, an antibody fragment, an antigen, a peptide, a polypeptide, a tissue-homing peptide, a small molecule, a synthetic polymer, a polymeric nanoparticle, a liposome, an enzyme substrate, a hormone, a neurotransmitter, a cellular metabolite, a viral particle, a viral capsid, a viral nanoparticle, a bacterial particle, a marker, a cell, a hapten, an avidin, streptavidin, monomeric streptavidin, biotin, a carbohydrate, an oligosaccharide, a polysaccharide, a nucleic acid, a deoxynucleic acid, a fragment of DNA, a fragment of RNA, an aptamer, a nucleotide triphosphate, an acyclo terminator triphosphate, or a PNA, or a combination thereof.

[0092] In some embodiments, the targeting molecule is a protein, peptide, or small molecule.

[0093] In some embodiments, the protein or peptide as the targeting molecule is selected from the group consisting of adrenocorticotropic hormone (ACTH), angiotensin II, atrial natriuretic factor (ANF), bombesin, bradykinin, brain-derived neurotrophic factor (BDNF), bone morphogenetic protein 2 (BMP-2), bone morphogenetic protein 6 (BMP-6), bone morphogenetic protein 7 (BMP-7), calcitonin, cardiotrophin 1 (BMP-2), CD22, CD40, cholecystokinin (CCK), ciliary neurotrophic factor (CNTF), CCL1-CCL28, CXCL1-CCL29, CXCL2-CCL30, CXCL1-CCL40, CXCL1-CCL50, CXCL1-CCL60, CXCL1-CCL70, CXCL1-CCL80, CXCL1-CCL90, CXCL1-CCL10, CXCL1-CCL12, CXCL1-CCL14, CXCL1-CCL16, CXCL1-CCL18, CXCL1-CCL19, CXCL1-CCL20, CXCL1-CCL21, CXCL1-CCL22, CXCL1-CCL23, CXCL1-CCL24, CXCL1-CCL25, CXCL1-CCL26, CXCL1-CCL27, CXCL1-CCL28, CXCL1-CCL29 ... XCL17, XCL1, XCL2, CX3CL1, Crypto-1 binding peptide, vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), endothelin-1, endothelin-1 / 3, FAS-ligand, fibroblast growth factor-1 (FGF-1), fibroblast growth factor-2 (FGF-2), fibroblast growth factor-4 (FGF-4), fibroblast growth factor-5 (FGF-5), fibroblast growth factor-6 (FGF-6), fibroblast growth factor-1 (FGF-7), fibroblast growth factor-1 (FGF-10), Flt-3, gastrin, gastrin-releasing peptide (GRP) ), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage-stimulating factor (GM-CSF), glucagon-like peptide-1 (GLP-1), hepatocyte growth factor (HGF), interferon alpha (IFN-a), interferon beta (IFN-b), interferon gamma (IFNg), insulin-like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), interleukin 1 (IL-1), interleukin 2 (IL-2), interleukin 3 (IL-3), interleukin 4 (IL-4), interleukin 5 (IL-5) -5), interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 9 (IL-9), interleukin 10 (IL-10), interleukin 11 (IL-11), interleukin 12 (IL-12), interleukin 13 (IL-13), interleukin 15 (IL-15), interleukin 17 (IL-17), interleukin 19 (IL-19), luteinizing hormone (LH), luteinizing-releasing hormone (LHRH), macrophage colony-stimulating factor (M-CSF),Monocyte chemotactic protein 1 (MCP-1), macrophage inflammatory protein 3a (MIP-3a), macrophage inflammatory protein 3b (MIP-3b), nerve growth factor (NGF), neuromedin B, neurotrophin 3 (NT-3), neurotrophin 4 (NT-4), neurotensin, neuropeptide Y, oxytocin, pituitary adenylate cyclase-activating peptide (PACAP), platelet-derived growth factor AA (PDGF-AA), platelet-derived growth factor AB (PDGF-AB), platelet-derived growth factor BB (PDGF-BB), platelet-derived growth factor Platelet-derived growth factor CC (PDGF-CC), platelet-derived growth factor DD (PDGF-DD), netrin-1 (NTN1), netrin-2 (NTN2), netrin-4 (NTN4), netrin-G1 (NTNG1) and netrin-G2 (NTNG2), ephrin-A1 (EFNA1), ephrin-A2 (EFNA2), ephrin-A3 (EFNA3), ephrin-A4 (EFNA4), ephrin-A5 (EFNA5), semaphorin 3A (SEMA3A), semaphorin 3B (SEMA3B), semaphorin 3C (SEMA3C), semaphorin 3D (SEMA3D), semaphorin 3 F (SEMA3F), semaphorin 3G (SEMA3G), semaphorin 4A (SEMA4A), semaphorin 4B (SEMA4B), semaphorin 4C (SEMA4C), semaphorin 4D (SEMA4D), semaphorin 4F (SEMA4F), semaphorin 4G (SEMA4G), semaphorin 5A (SEMA5A), semaphorin 5B (SEMA5B), semaphorin 6A (SEMA6A), semaphorin 6B (SEMA6B), semaphorin 6D (SEMA6D), semaphorin 7A (SEMA7A), SLIT1, SLIT2, SLIT3, SLIT and NTRK-like family member 1 (SLITRK1), SLIT and NTRK-like family member 2 (SLITRK2), SLIT and NTRK-like family member 3 (SLITRK3), SLIT and NTRK-like family member 4 (SLITRK4), SLIT and NTRK-like family member 5 (SLITRK5), SLIT and NTRK-like family member 6 (SLITRK6), prostaglandin E2 (PGE2), RANTES, somatostatin-14, somatostatin-28, stem cell factor (SCF),The polypeptide is selected from stromal cell-derived factor 1 (SDF-1), substance P, thyroid-stimulating hormone (TSH), transforming growth factor alpha (TGF-α), transforming growth factor beta (TGF-b), tumor necrosis factor alpha (TNF-α), thrombin, vasoactive intestinal peptide (VIP), Wntl, Wnt2, Wnt2b / 13, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt7c, Wnt8, Wnt8a, Wnt8b, Wnt8c, Wntl0a, Wntl0b, Wnt11, Wnt14, Wnt15, or Wnt16, sonic hedgehog, desert hedgehog, and Indian hedgehog.

[0094] In some embodiments, the small molecule targeting molecule is selected from the group consisting of a VEGFR inhibitor, a TNFR1 inhibitor, a growth factor receptor inhibitor, and combinations thereof. For example, the small molecule VEGFR inhibitor may be selected from the group consisting of, but not limited to, pazopanib, semaxinib, axitinib, cabozantinib, aflibercept, brivanib, tivozanib, ramucirumab, motesanib, vatalanib, cediranib, and combinations thereof.

[0095] In some embodiments, the targeting molecule is an antibody or antibody fragment.

[0096] In some embodiments, the antibody is selected from the group consisting of cetuximab, panitumumab, zalutumumab, nimotuzumab, trastuzumab, Ado-trastuzumab emtansine, tositumomab (Bexxar®), rituximab (Rituxan, Mabthera), ibritumomab tiuxetan (Zevalin), daclizumab (Zenapax), gemtuzumab (Mylotarg), alemtuzumab, CEA-scan Fab fragment, OC125 monoclonal antibody, ab75705, B72.3, bevacizumab (Avastin®), afatinib, axitinib, bosutinib, cabozantinib, ceritinib, crizotinib, dabrafenib, dasatinib, erlotinib, everolimus, ibrutinib, imatinib, lapatinib, lenvatinib, nilotinib, olaparib, palbociclib, pazopanib, pertuzumab mab, ramucirumab, regorafenib, ruxolitinib, sorafenib, sunitinib, temsirolimus, trametinib, vandetanib, vemurafenib, vismodegib, basiliximab, ipilimumab, nivolumab, pembrolizumab, MPDL3280A, pidilizumab (CT-011), AMP-224, MSB001078C, or MEDI4736, or an antigen-binding antibody fragment thereof.

[0097] In some embodiments, the antibody is cetuximab, panitumumab, trastuzumab, BMS-935559, MEDI4736, MPDL3280A, or MSB0010718C, or an antigen-binding antibody fragment thereof.

[0098] Antibody fragments include, but are not limited to, antigen-binding antibody fragments that are F(ab) fragments, F(ab') fragments, VHH fragments (single domain antibodies, also called nanobodies), single-chain variable fragments (scFv), multivalent scFv, bispecific scFv, or scFv-CH3 dimers.

[0099] In some embodiments, methods for producing a drug containing a phthalocyanine dye represented by the above formula (I) bound or conjugated to a targeting molecule are known, and reference can be made, for example, to WO2015 / 187677, WO2017 / 031363, JP 2019-218374, etc.

[0100] A drug in which a phthalocyanine dye represented by formula (I), which is a water-soluble phthalocyanine silicon complex, is bound or conjugated to a targeting molecule is useful as a new photoimmunotherapeutic drug.

[0101] In some embodiments, the agent containing the phthalocyanine dye represented by the above formula (I) bound or conjugated to a targeting molecule is an agent for inducing cytotoxicity by apoptosis in target cells.

[0102] In some embodiments, the target cell is a stem cell, a proliferative cell, a cancer cell, a hyperplastic cell, a tumor cell, an inflammatory cell, a neuronal cell, or a cell infected with a pathogen, hi some embodiments, the target cell is a cancer cell, a tumor cell, an inflammatory cell, or a neuronal cell.

[0103] In some embodiments, the induction of cytotoxicity is brought about by administering the agent to the tumor cells and irradiating the tumor cells with light after administering the agent.

[0104] Induction of cytotoxicity through apoptosis in target cells using agents containing phthalocyanine dyes bound or conjugated to targeting molecules is known, see, for example, WO2015 / 187677, WO2017 / 031363, JP 2019-218374, etc.

[0105] Cytotoxicity is induced by (1) administering to a subject or cells of a subject in vivo or in vitro a therapeutically effective amount of a drug containing a phthalocyanine dye of formula (I) conjugated to a targeting molecule that specifically binds to target cells of the subject, and (2) irradiating the cells with a suitable excitation light in an amount effective to induce cell death by apoptosis.

[0106] In some embodiments, the control is a subject suffering from a disease or condition, wherein the disease or condition is selected from the group consisting of (i) vascular disease, cancer, bacterial biofilm-induced infection, antibiotic-resistant wound infection, actinic keratosis, rosacea, acne, psoriasis, and wet age-related macular degeneration, or (ii) the disease or condition is selected from the group consisting of breast cancer, colorectal cancer, esophageal cancer, lung cancer, prostate cancer, cervical cancer, ovarian cancer, gastric cancer, pancreatic cancer, liver cancer, bladder cancer, brain cancer, head and neck cancer, cancer of the neuroendocrine system, skin cancer, and combinations thereof.

[0107] In some embodiments, the subject is a subject who has or has had a tumor, particularly a solid tumor. Thus, a drug containing the phthalocyanine dye represented by the above formula (I) bound or conjugated to a targeting molecule can be used to treat the tumor in the subject.

[0108] In some embodiments, the target cell is a stem cell, a proliferative cell, a cancer cell, a hyperplastic cell, a tumor cell, an inflammatory cell, a neuronal cell, or a cell infected with a pathogen, hi some embodiments, the target cell is a cancer cell, a tumor cell, an inflammatory cell, or a neuronal cell.

[0109] Suitable excitation light has a wavelength between 660 and 740 nm.

[0110] Next, a method for producing the phthalocyanine dye represented by formula (I) will be described with reference to an embodiment of a synthetic scheme for an illustrative compound. Compounds in which a linker is bonded to a phthalocyanine skeleton at the ortho position are known, as described in WO 2004 / 038378 A2, WO 2015 / 187677 A1, etc.

[0111] In contrast, in an embodiment of the present invention, 4-nitrophthalonitrile is used as a starting material and reacted with a hydroxy-substituted alkyne having a carbon-carbon triple bond at one end and a hydroxy group at the other end to synthesize a phthalonitrile compound (1) substituted with a nitro group at the 4-position. This compound is then mixed with sodium methoxide in an ammonium methanol solution to produce diiminoisoindoline (2).

[0112]

[0113]

[0114] Examples of hydroxy-substituted alkynes include the following:

[0115]

[0116] Instead of the hydroxy-substituted alkyne, a thiol-substituted alkyne having a carbon-carbon triple bond at one end and a thiol group at the other end can also be used.

[0117] Next, diiminoisoindoline (2) is added to quinoline and reacted with 1,3-diiminoisoindoline in the presence of SiCl to obtain crude phthalocyanine compound (3). Instead of quinoline, a basic, high-boiling solvent such as tributylamine can also be used. The reaction temperature is preferably 150°C to 220°C (reflux), and the reaction time is preferably 1 hour or more but less than 18 hours.

[0118]

[0119] Thereafter, by a well-known method such as that described in WO 2004 / 038378 A2 and WO 2015 / 187677 A1, the ligand bonded to the silicon of the phthalocyanine ring of compound (3) is bonded to the phthalocyanine ring via a siloxane bond (—Si—O—Si—), converting it into a ligand containing a water-soluble substituent, thereby obtaining compound (6).

[0120]

[0121]

[0122]

[0123] Next, using the azide-alkyne click reaction (azide-alkyne cycloaddition reaction), the carbon-carbon bond at the end of the functional group attached to the phthalocyanine ring of compound (6) is reacted with an azide under copper catalysis to form compound (7) with a linker containing a triazolyldiyl group. The azide to be reacted with compound (6) is designed so that the linker of compound (7) has a reactive group at its end.

[0124]

[0125] As the azide, an azide having a heteroatom in the molecule as described below may be used.

[0126] Compound (7) can also be converted to compound (8), in which a reactive group more suitable for attaching a dye to a targeting molecule is introduced at the end of the linker (see the example below for NHS conversion using di(succinimidyl) carbonate).

[0127]

[0128] According to the method for producing a phthalocyanine dye of this embodiment of the present invention, the bonding position of the linker to the phthalocyanine ring is changed from the ortho position to the meta position, thereby alleviating steric hindrance in the synthesis of the skeleton, and the number of steps required from the starting material 4-nitrophthalonitrile to compound (8) is reduced to eight. Furthermore, the linker can be easily derivatized by elongating it using click chemistry.

[0129] The method for producing a phthalocyanine dye of the present invention is characterized by utilizing click chemistry, and therefore includes the following production methods (1) to (5) in addition to the production methods described above.

[0130] (1) Instead of synthesizing the phthalonitrile compound (1) in which the nitro group at the 4-position is substituted by a hydroxy-substituted alkyne having a terminal hydroxy group with the starting material 4-nitrophthalonitrile, an azide having a hydroxy or thiol group (e.g., N3-R 1 -OH or N3-R 1 -SH, R 1 is a direct bond, C1-C optionally interposed with a heteroatom 10 In the formula below, R may be an alkylene, polyethylene glycol (PEG), phenylene, ester, amide, carbamate, or a combination of two or more thereof. 1is -CH2-CH2-) to synthesize a phthalonitrile compound (1)' substituted with a nitro group at the 4-position. Also, instead of reacting the carbon-carbon bond at the terminal of the functional group bonded to the phthalocyanine ring of compound (6) with an azide to form compound (7) having a linker containing a triazolyldiyl group, the azide group at the terminal of the functional group bonded to the phthalocyanine ring of compound (6)' can be substituted with a compound having a carbon-carbon triple bond (e.g., R 1 -C≡CR 2 -COOX, R 1 is hydrogen or any other functional group, and R 2 is a direct bond, C1-C optionally interposed with a heteroatom 10 alkylene, PEG, phenylene, ester, amide, carbamate, or a combination of two or more thereof, and X is hydrogen or Na. 1 is hydrogen, R 2 is -CH2-CH2-CH2-, X is hydrogen) to form compound (7)' having a linker containing a triazolyldiyl group. In this case, too, the phthalocyanine dye of the present invention can be produced by utilizing the azide-alkyne click reaction.

[0131] (2) The starting material, 4-nitrophthalonitrile, is reacted with a strained alkyne bearing a hydroxyl or thiol group. The cycloaddition reaction between azides and strained alkynes is well known as a copper-free click reaction (PNAS 104 (43) 16793-16797. DOI: 10.1073 / pnas.0707090104).

[0132] Examples of strained alkynes include cyclooctyne derivatives, dibenzocyclooctyne derivatives, diazacyclononyne derivatives, etc. Note that cyclooctyne derivatives, dibenzocyclooctyne derivatives, and diazacyclononyne derivatives refer to compounds having a cyclooctyne ring, a dibenzocyclooctyne ring, and a diazacyclononyne ring, respectively, in which at least one hydrogen atom on a carbon atom of the ring is substituted with another functional group.

[0133] Examples of cyclooctyne derivatives include cyclooctyne derivatives represented by formula (12). Examples of dibenzocyclooctyne derivatives include dibenzocyclooctyne derivatives represented by formula (13). Examples of compounds having a diazacyclononyne ring include diazacyclononyne derivatives represented by formula (14).

[0134]

[0135] In formula (12), R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are each independently hydrogen or any other substituent, provided that R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least one of the groups has a hydroxy group or a thiol group.

[0136]

[0137] In formula (13), R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 are each independently hydrogen or any other substituent.

[0138] R 9 is C1-C optionally interrupted by hydrogen, a heteroatom or an ester; 10 Hydroxy C1-C optionally interrupted by alkyl, heteroatom or ester 10 Thiol C1-C optionally interrupted by alkyl, heteroatom or ester 10 It is an alkyl, ester, or amide.

[0139] However, R 1 , R 2 , R 3 , R 4 , R5 , R 6 , R 7 , R 8 , R 9 At least one of the groups has a hydroxy group or a thiol group.

[0140]

[0141] In formula (14), Y is p-toluenesulfonyl, methanesulfonyl, nitrobenzenesulfonyl, succinoyl, maleimidoalkyl, or -HCl.

[0142] Z is p-toluenesulfonyl, methanesulfonyl, nitrobenzenesulfonyl, succinoyl, maleimidoalkyl, or -HCl.

[0143] Y and Z may be the same or different.

[0144] R 1 , R 2 , R 3 , R 4 , and R 5 are each independently hydrogen, C1-C6 alkyl, or hydroxy or thiol, provided that R 1 , R 2 , R 3 , R 4 , and R 5 One of the groups is hydroxy or thiol.

[0145] (3) Instead of synthesizing the phthalonitrile compound (1) in which the nitro group at the 4-position is substituted by a hydroxy-substituted alkyne having a terminal hydroxy group with the starting material 4-nitrophthalonitrile, an azide having a hydroxy or thiol group (e.g., N3-R 1 -OH, R 1 is a direct bond, C1-C optionally interposed with a heteroatom 10 In the formula below, R may be an alkylene, polyethylene glycol (PEG), phenylene, ester, amide, carbamate, or a combination of two or more thereof. 1is -CH2-CH2-) to synthesize phthalonitrile compound (1)' substituted with a nitro group at the 4-position. Also, instead of reacting the carbon-carbon bond at the end of the functional group bonded to the phthalocyanine ring of compound (6) with an azide to form compound (7) having a linker containing a triazolyldiyl group, compound (6)' is reacted with a strained alkyne having a carboxy group to form a compound having a linker containing a cyclooctyne ring, a dibenzocyclooctyne ring, or a diazacyclononyne ring.

[0146] Examples of strained alkynes include cyclooctyne derivatives, dibenzocyclooctyne derivatives, and diazacyclononyne derivatives. Examples of cyclooctyne derivatives include the cyclooctyne derivative represented by formula (15). Examples of dibenzocyclooctyne derivatives include the dibenzocyclooctyne derivative represented by formula (16). Examples of diazacyclononyne derivatives include the diazacyclononyne derivative represented by formula (17).

[0147]

[0148] In formula (15), R 1 , R 2 , R 4 , R 5 and R 6 are each independently hydrogen or any other substituent. 3 is hydrogen or any other functional group.

[0149] R 3 is a direct bond, C1-C optionally interposed with a heteroatom 10 It may be alkylene, PEG, phenylene, ester, amide, carbamate, or a combination of two or more thereof.

[0150] X is hydrogen or Na.

[0151]

[0152] In formula (16), R 1 , R 2 , R 3 , R 4 , R 5 , R6 , R 7 and R 8 are each independently hydrogen or any other substituent.

[0153] R 9 is hydrogen, a direct bond, or C1-C optionally interrupted by a heteroatom. 10 It may be alkylene, PEG, phenylene, ester, amide, carbamate, or a combination of two or more thereof.

[0154] X is hydrogen or Na.

[0155]

[0156] In formula (17), Y can be p-toluenesulfonyl, methanesulfonyl, nitrobenzenesulfonyl, succinoyl, maleimidoalkyl, or H-HCl.

[0157] Z can be p-toluenesulfonyl, methanesulfonyl, nitrobenzenesulfonyl, succinoyl, maleimidoalkyl, or H-HCl.

[0158] Y and Z may be the same or different.

[0159] R 1 , R 2 , R 3 , R 4 , and R 5 At least one of them is -R 6 R is a group represented by -COOX 6 is hydrogen, a direct bond, or C1-C optionally interrupted by a heteroatom. 10 alkylene, PEG, phenylene, ester, amide, carbamate, or a combination of two or more thereof, and X is hydrogen or Na.

[0160] R 1 , R 2 , R 3 , R 4 , and R 5 The remaining groups are each independently hydrogen or any other substituent.

[0161] Again, the copper-free strain-promoted azide-alkyne click reaction can be utilized to prepare the phthalocyanine dyes of the present invention.

[0162] In some embodiments, R 3 Ga-R 6 -COOX, and R 1 , R 2 , R 4 , and R 5 are each independently hydrogen or any other substituent.

[0163] (4) The starting material, 4-nitrophthalonitrile, is reacted with a strained alkene or strained alkyne. Alternatively, instead of reacting compound (6) with an azide, a tetrazine derivative is used. The inverse electron-demand Diels-Alder cycloaddition reaction of tetrazine with a strained alkene and the inverse electron-demand Diels-Alder cycloaddition reaction of tetrazine with a strained alkyne are well known (J. Am. Chem. Soc. 2008, 130, 41, 13518-13519, DOI: 10.1021 / ja8053805, Chem Soc Rev 2013; 42: 5131-42. DOI: 10.1039 / c3cs60049a).

[0164] Examples of strained alkenes include, but are not limited to, cyclopropene derivatives, trans cyclooctene derivatives, and novonornene derivatives. Examples of cyclopropene derivatives include cyclopropene derivatives represented by formula (18). Examples of trans cyclooctene derivatives include trans cyclooctene derivatives represented by formula (19). Examples of novonornene derivatives include novonornene derivatives represented by formula (20).

[0165]

[0166] In formula (18), R 1 is a direct bond, C1-C optionally interposed with a heteroatom 10It may be an alkylene, polyethylene glycol (PEG), phenylene, ester, amide, carbamate, or a combination of two or more thereof.

[0167]

[0168] In formula (19), R 1 is a direct bond, C1-C optionally interposed with a heteroatom 10 It may be an alkylene, polyethylene glycol (PEG), phenylene, ester, amide, carbamate, or a combination of two or more thereof.

[0169]

[0170] In formula (20), R 1 is a direct bond, C1-C optionally interposed with a heteroatom 10 It may be an alkylene, polyethylene glycol (PEG), phenylene, ester, amide, carbamate, or a combination of two or more thereof.

[0171] Examples of strained alkynes include cyclooctyne derivatives, dibenzocyclooctyne derivatives, and diazacyclononyne derivatives. Examples of cyclooctyne derivatives include the cyclooctyne derivative represented by the above formula (12). Examples of dibenzocyclooctyne derivatives include the dibenzocyclooctyne derivative represented by the above formula (13). Examples of azacyclononyne derivatives include the diazacyclononyne derivative represented by the above formula (14).

[0172] The tetrazine derivative is, for example, a compound represented by the following formula (21).

[0173]

[0174] In formula (21), R 1 is hydrogen or any other functional group.

[0175] R 2 is a direct bond, C1-C optionally interposed with a heteroatom 10 It may be alkylene, PEG, phenylene, ester, amide, carbamate, or a combination of two or more thereof.

[0176] X is hydrogen or Na.

[0177] The reaction of tetrazine with a strained alkene or strained alkyne forms a linker containing a fused diazine and cycloalkane ring, such as that shown in formula (22).

[0178]

[0179] In formula (22), ring A is a C3-C 10 It is cycloalkyl.

[0180] (5) Instead of synthesizing the phthalonitrile compound (1) having a nitro group substituted at the 4-position by reacting the starting material 4-nitrophthalonitrile with a hydroxy-substituted alkyne having a terminal hydroxy group, a tetrazine derivative having a hydroxy or thiol group is reacted. Also, instead of reacting the carbon-carbon bond at the terminal of the functional group bonded to the phthalocyanine ring of compound (6) with an azide to form compound (7) having a linker containing a triazolyldiyl group, a strained alkene having a carboxy group or a carboxylate or a strained alkyne having a carboxy group or a carboxylate is reacted to form a compound having a linker containing a condensed ring of a diazine and a cycloalkane.

[0181] The tetrazine derivative having a hydroxy group or a thiol group is, for example, a tetrazine derivative represented by formula (23).

[0182]

[0183] In formula (23), R 1 is hydrogen or any other functional group.

[0184] R 2 is a direct bond, C1-C optionally interposed with a heteroatom 10 It may be an alkylene, polyethylene glycol (PEG), phenylene, ester, amide, carbamate, or a combination of two or more thereof.

[0185] X is oxygen or sulfur.

[0186] Examples of strained alkenes include, but are not limited to, cyclopropene derivatives, trans cyclooctene derivatives, and novonornene derivatives. Examples of cyclopropene derivatives include cyclopropene derivatives represented by formula (24). Examples of trans cyclooctene derivatives include trans cyclooctene derivatives represented by formula (25). Examples of novonornene derivatives include novonornene derivatives represented by formula (26).

[0187]

[0188] In formula (24), R 1 is a direct bond, C1-C optionally interposed with a heteroatom 10 It may be alkylene, PEG, phenylene, ester, amide, carbamate, or a combination of two or more thereof.

[0189] X is hydrogen or Na.

[0190]

[0191] In formula (25), R 1 is a direct bond, C1-C optionally interposed with a heteroatom 10 It may be alkylene, PEG, phenylene, ester, amide, carbamate, or a combination of two or more thereof.

[0192] X is hydrogen or Na.

[0193]

[0194] In formula (26), R 1 is a direct bond, C1-C optionally interposed with a heteroatom 10 It may be alkylene, PEG, phenylene, ester, amide, carbamate, or a combination of two or more thereof.

[0195] X is hydrogen or Na.

[0196] Examples of strained alkynes include cyclooctyne derivatives, dibenzocyclooctyne derivatives, and diazacyclononyne derivatives. Examples of cyclooctyne derivatives include the cyclooctyne derivative represented by the above formula (15). Examples of dibenzocyclooctyne derivatives include the dibenzocyclooctyne derivative represented by the above formula (16). Examples of diazacyclononyne derivatives include the diazacyclononyne derivative represented by the above formula (17).

[0197] The reaction of tetrazine with a strained alkene or strained alkyne forms a linker containing a fused diazine and cycloalkane ring, such as that shown in formula (22).

[0198]

[0199] In formula (22), ring A is a C3-C 10 It is cycloalkyl.

[0200] In the following description, any of the above click reactions (1) to (5) may be applied instead of the azide-alkyne reaction.

[0201] According to another aspect of the present invention, there is provided a method for producing a phthalocyanine dye represented by formula (IV), which comprises reacting a compound represented by formula (III) with reagent (B) to obtain a compound represented by formula (IV).

[0202] (In formulas (III) and (IV), M is selected from Si, Ge, Sn, or Al; R 2 , R 3 , R 7 , and R 8 are each independently selected from optionally substituted alkyl and optionally substituted aryl; R 4 , R 5 , R 6 , R 9 , R 10 , and R 11are each independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkanoyl, optionally substituted alkoxycarbonyl, optionally substituted alkylcarbamoyl, and a chelating ligand, wherein R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 at least one of R contains a water-soluble group; 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 and R 23 are each independently selected from hydrogen, halogen, optionally substituted alkylthio, and optionally substituted alkylamino; Y is selected from a direct bond, oxygen, sulfur, or optionally substituted nitrogen; X 1 and X 2 each independently represents a C-C group optionally interrupted by a heteroatom; 10 is alkylene, and X 3 and X 4 each independently represents a C-C group optionally interrupted by a heteroatom; 10 selected from alkylene, polyethylene glycol (PEG), phenylene, ester, amide, carbamate, or a combination of two or more thereof, Z is an optionally substituted triazolyldiyl group or a fused ring of an optionally substituted diazine and a cycloalkane, P 1is selected from among an amine-reactive chemical group, a sulfhydryl-reactive chemical group, an activated ester, an acyl halide, an alkyl halide, an acid anhydride group, a carboxy, a carboxylate, a carboxylate, a carbodiimide, a carbonate, a carbamate, a haloacetamide, an isocyanate, an isothiocyanate, a maleimide, an N-hydroxysuccinimide ester, a phosphoramidite, a platinum complex, a sulfonate ester, and a thiocyanate; A has a first functional group and reagent (B) has a second functional group that reacts with the first functional group of A via a Click reaction.

[0203] The combination of the first functional group and the second functional group that undergo the Click reaction can be selected by one skilled in the art.

[0204] In some embodiments, A and reagent (B) are any of the following combinations (i) to (vi): (i) A is a group having a carbon-carbon triple bond (e.g., an alkynyl group), and reagent (B) is an azide; (ii) A is an azide group, and reagent (B) is a compound having a carbon-carbon triple bond (e.g., an alkyne); (iii) A is a group having a cyclooctyne ring, a group having a dibenzocyclooctyne ring, or a group having a diazacyclononyne ring, and reagent (B) is an azide; or (iv) A is an azide group, and reagent (B) is a strained alkyne (e.g., a cyclooctyne derivative, a dibenzocyclooctyne derivative, or a diazacyclononyne derivative). (v) A is a group having a group resulting from the abstraction of one or more hydrogen atoms from a strained alkene (e.g., a group having a cyclopropenyl group, a group having a transcyclooctenyl group, or a group having a novonornene group) or a group having a group resulting from the abstraction of one or more hydrogen atoms from a strained alkyne (e.g., a group having a cyclooctyne ring, a group having a dibenzocyclooctyne ring, or a group having a diazacyclononyne ring), and reagent (B) is a tetrazine derivative. (vi) A is a group having a tetrazinyl group, and reagent (B) is a strained alkene (e.g., a cyclopropene derivative, a transcyclooctene derivative, or a novonornene derivative) or a strained alkyne (e.g., a cyclooctyne derivative, a dibenzocyclooctyne derivative, or a diazacyclononyne derivative). According to another aspect of the present invention, there is provided a method for producing a phthalocyanine dye represented by formula (VII), which comprises reacting a compound represented by formula (VI) with an azide (X) to obtain a compound represented by formula (VII).

[0205]

[0206] In formulas (VI), (VII), and (X), M is selected from Si, Ge, Sn, or Al; R 2 , R 3 , R 7 , and R 8 are each independently selected from optionally substituted alkyl and optionally substituted aryl; R 4 , R 5 , R 6 , R 9 , R10 , and R 11 are each independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkanoyl, optionally substituted alkoxycarbonyl, optionally substituted alkylcarbamoyl, and a chelating ligand, wherein R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 at least one of R contains a water-soluble group; 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 and R 23 are each independently selected from hydrogen, halogen, optionally substituted alkylthio, and optionally substituted alkylamino; Y is selected from a direct bond, oxygen, sulfur, or optionally substituted nitrogen; X 1 and X 2 each independently represents a C-C group optionally interrupted by a heteroatom; 10 is alkylene, and X 3 and X 4 each independently represents a C-C group optionally interrupted by a heteroatom; 10 alkylene, polyethylene glycol (PEG), phenylene, ester, amide, carbamate, or a combination of two or more thereof; Z is an optionally substituted triazolyldiyl group; P 1 is selected from among amine-reactive chemical groups, sulfhydryl-reactive chemical groups, activated esters, acyl halides, alkyl halides, acid anhydride groups, carboxy, carboxylate, carboxylate, carbodiimide, carbonate, carbamate, haloacetamide, isocyanate, isothiocyanate, maleimide, N-hydroxysuccinimide ester, phosphoramidite, platinum complex, sulfonate ester, and thiocyanate.

[0207] In some embodiments, the azide (X) is P 2 -X 4 - Expressed as N3. P 2 reacts with a compound of formula (VI), such as a carboxylate or carboxylate salt, to form P 1 is a reactive functional group that forms

[0208] In some embodiments, the compound of formula (IV) may include a P 1 a compound having a functional group reactive with a group having a reactive functional group to obtain a compound represented by the following formula (I), or a compound having a functional group reactive with a group having a reactive ... 1 and reacting a group with a compound having a reactive functional group to obtain a compound represented by the following formula (I):

[0209]

[0210] (In formula (I), M is selected from Si, Ge, Sn, or Al, L is a group represented by the following formula (II), -X 4 -ZX 3 -Y- Formula (II) (wherein Y is selected from a direct bond, oxygen, sulfur, or optionally substituted nitrogen; X 3 and X 4 each independently represents a C-C group optionally interrupted by a heteroatom; 10 R is a group selected from alkylene, polyethylene glycol (PEG), phenylene, ester, amide, carbamate, or a combination of two or more thereof; Z is an optionally substituted triazolyldiyl group or an optionally substituted fused ring of diazine and cycloalkane; Q is a reactive group for attachment of the dye to a targeting molecule; 2 , R 3 , R 7 , and R 8 are each independently selected from optionally substituted alkyl and optionally substituted aryl; R 4 , R 5 , R 6 , R9 , R 10 , and R 11 are each independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkanoyl, optionally substituted alkoxycarbonyl, optionally substituted alkylcarbamoyl, and a chelating ligand, wherein R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 at least one of R contains a water-soluble group; 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 and R 23 are each independently selected from hydrogen, halogen, optionally substituted alkylthio, and optionally substituted alkylamino, and X 1 and X 2 each independently represents a C-C group optionally interrupted by a heteroatom; 10 It is alkylene.

[0211] In some embodiments, P of formula (VII) 1 The group is carboxy, carboxylate, or carboxylate, and P of formula (VII) 1 The compound having a functional group reactive with the group is di(succinimidyl) carbonate.

[0212] In some embodiments, any one of compounds (iii) to (vi) below is provided.

[0213]

[0214] (wherein M is selected from Si, Ge, Sn, or Al; R 12 , R 13 , R 14 , R 15 , R 16 , R17 , R 18 , R 19 , R 20 , R 21 , R 22 and R 23 are each independently selected from hydrogen, halogen, optionally substituted alkylthio, and optionally substituted alkylamino; Y is selected from a direct bond, oxygen, sulfur, or optionally substituted nitrogen; and X 3 is a C1-C alkyl group optionally interrupted by a heteroatom. 10 alkylene, polyethylene glycol (PEG), phenylene, ester, amide, carbamate, or a combination of two or more thereof.

[0215]

[0216] (In formula (iv), M is selected from Si, Ge, Sn, or Al; R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 and R 23 are each independently selected from hydrogen, halogen, optionally substituted alkylthio, and optionally substituted alkylamino; and R 25 are each independently C1-C 10 an alkyl group, Y is selected from a direct bond, oxygen, sulfur, or optionally substituted nitrogen, and X 3 is a C1-C alkyl group optionally interrupted by a heteroatom. 10 alkylene, polyethylene glycol (PEG), phenylene, ester, amide, carbamate, or a combination of two or more thereof.

[0217]

[0218] (wherein M is selected from Si, Ge, Sn, or Al; R 2 , R 3, R 7 , and R 8 are each independently selected from optionally substituted alkyl and optionally substituted aryl; R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 and R 23 are each independently selected from hydrogen, halogen, optionally substituted alkylthio, and optionally substituted alkylamino; Y is selected from a direct bond, oxygen, sulfur, or optionally substituted nitrogen; X 1 and X 2 each independently represents a C-C group optionally interrupted by a heteroatom; 10 alkylene, and X 3 each independently represents a C-C group optionally interrupted by a heteroatom; 10 alkylene.)

[0219]

[0220] (wherein M is selected from Si, Ge, Sn, or Al; R 2 , R 3 , R 7 , and R 8 are each independently selected from optionally substituted alkyl and optionally substituted aryl; R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 are each independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkanoyl, optionally substituted alkoxycarbonyl, optionally substituted alkylcarbamoyl, and a chelating ligand, wherein R 4 , R 5 , R 6 , R 9 , R 10 , and R11 at least one of R contains a water-soluble group; 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 and R 23 are each independently selected from hydrogen, halogen, optionally substituted alkylthio, and optionally substituted alkylamino; Y is selected from a direct bond, oxygen, sulfur, or optionally substituted nitrogen; X 1 and X 2 each independently represents a C-C group optionally interrupted by a heteroatom; 10 alkylene, and X 3 each independently represents a C-C group optionally interrupted by a heteroatom; 10 alkylene.

[0221] In some specific embodiments, the compounds (iii) to (iv) are any of the following compounds 3 to 6, respectively.

[0222]

[0223] The disclosures of all patent applications and publications cited herein are hereby incorporated by reference in their entirety.

[0224] The following examples are intended for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Unless otherwise specified, reagents are commercially available or are obtained or prepared according to conventional techniques or literature procedures in the art.

[0225] Synthesis Example 1: Synthesis of Compound 8 (1) Synthesis of Compound 1: 3-Butyn-1-ol (5 mL) was added dropwise to a solution of 4-nitrophthalonitrile (4.89 g, 28.2 mmol) and potassium carbonate (19.1 g, 191 mmol) in DMSO (50 mL) and stirred at 50°C for 18 hours. After cooling to room temperature, the mixture was diluted with purified water (500 mL). The residue after filtration was washed with purified water and methanol. The residue was collected and dried to obtain Compound 1 (2.72 g, 18.7 mmol, 49.2%). 1 H NMR (500 MHz, CDCl3): δ 7.72 (d, J =8.9 Hz, 1H), 7.29 (d, J = 2.6 Hz. 1H), 7.22 (dd, J = 8.9, 2.6 Hz, 1H), 4.18 (t, J = 6.7 Hz, 2H), 2.73 (td, J = 6.7, 2.7 Hz, 2H), 2.06 (t, J = 2.7 Hz, 1H) 13 C NMR (126 MHz, CDCl3); δ 161.2, 135.4, 119.8, 119.5, 117.5, 115.7, 115.3, 107.7, 79.3, 70.8, 67.1, 19.4 LRMS (ESI + ) m / z: [M+H + ] calcd for C 12 H8N2O, 197; found, 197

[0226]

[0227] (2) Synthesis of Compound 2 Compound 1 (1.41 g, 196 mmol) and sodium methoxide (21.0 mg, 0.400 mmol) were dissolved in 7N ammonium hydroxide in methanol (20 mL) and stirred at 50°C for 18 hours under an argon atmosphere. After concentration on a rotary evaporator, the solution was purified by silica gel column chromatography (gradient: 10% for 8 min, 10-58% for 3 min; 58% for 2 min; 58-100% for 1 min; 100% for 45 min with CHOH in CHCl). 2,Compound 2 (1.26 g, 52.9 mmol, 82.1%) was obtained by MPLC (Yamazen Corporation Universal Column premium) (30 μm, 2.3 × 12.3 cm, flow rate 10 ml / min). 1 H NMR (500 MHz, DMSO-d6): δ 8.44 (brs, 3H), 7.70 (d, J = 8.2 Hz), 7.43 (brs, 1H), 7.24 (dd, J = 8.2, 2.3 Hz, 1H), 4.15 (t, J= 6.6 Hz, 2H), 2.91 (t, J = 2.7 Hz, 1H), 2.68 (td, J = 6.6, 2.7 Hz, 2H) 13 CNMR (126 MHz, DMSO-d-6); δ 169.2, 160.1, 138.8, 128.8, 122.3, 116.7, 107.0, 81.2, 72.6, 66.3, 0.334, LRMS (ESI + ) m / z: [M+H + ] calcd for C 12 H 11 N3O, 214; found, 214

[0228]

[0229] (3) Synthesis of Compound 3 Compound 2 (680 mg, 3.2 mmol) and 1,3-diiminoisoindoline (870 mg, 6.0 mmol) were added to quinoline (8 mL) and stirred at room temperature for 10 minutes under an argon atmosphere. Tetrachlorosilane (1 mL) was added dropwise to this solution, and the mixture was stirred at 200 °C for 2 hours. After cooling to room temperature, the mixture was diluted with purified water (100 mL), and the blue precipitate was filtered. This precipitate was washed with purified water and methanol and dried under vacuum to obtain crude compound 3.

[0230]

[0231] (4) Synthesis of Compound 4: The total amount of compound 3 obtained in (3) above was dissolved in anhydrous pyridine (20 mL) and anhydrous n-butanol (20 mL), and the solution was stirred at 110 °C for 18 hours under an argon atmosphere and protected from light. After concentration using a rotary evaporator, crude compound 4 was obtained by column chromatography (gradient: 10% for 15 min; 10-28% for 18 min; 28-100% for 8 min CHCl in hexane, MCLP (Yamazen Corporation Universal Column Premium Amino) (30 μm, 2.3 × 12.3 cm), flow rate 10 mL / min).

[0232]

[0233] (5) Synthesis of Compound 5: The entire amount of compound 4 obtained in (4) above was dissolved in dehydrated pyridine (15 mL). 3-Aminopropyldimethylethoxysilane (3.5 mL) was added dropwise under stirring under an argon atmosphere, and the mixture was then stirred at 110 °C for 18 hours in the dark. After concentration using a rotary evaporator, crude compound 5 was obtained by column chromatography (Gradient: 1% for 8 min; 1-3% for 10 min; 3% for 7 min; 10-20% for 15 min; 20% for 7 min CHOH in CHCl, MCLP (Yamazen Corporation Universal Column Amino Premium) (30 μm, 2.3 × 12.3 cm), flow rate 10 mL / min).

[0234]

[0235] (6) Synthesis of Compound 6. To the entire amount of compound 5 obtained in (5) above, dehydrated methanol (15 mL), diisopropylethylamine (6 mL), and 1,3-propane sultone (3 mL) were added. After freeze-degassing, the mixture was stirred at 50 °C for 18 hours under an argon atmosphere, shielded from light. After concentration using a rotary evaporator, the mixture was diluted with purified water / acetonitrile (9:1) and subjected to reverse-phase column chromatography (gradient: 10% for 8 min; 10-80% for 40 min; 80-100% for 10 min; 100% for 10 min; CHCN containing 100 mM triethylammonium acetate (TEAA) in HO containing 100 mM TEAA, Sfaer C18 D-Duo 100Å 30 μm, flow rate 12 mL / min, Sfaer's "ae" is an umlaut, but this is used as an alternative notation). Then, crude compound 6 (870 mg) was obtained by reverse-phase column chromatography (Gradient: 1% for 7 min; 1-100% for 1.5 min, 100% for 4.2 min CHOH in HO, MPLC (Sfaer C18 D-Duo 100Å 30 μm), flow rate 12 mL / min).

[0236]

[0237] (7) Synthesis of Compound 7. 5-Azidovaleric acid (42.7 mg, 0.30 mmol) and sodium bicarbonate (33.1 mg, 0.39 mmol) were dissolved in purified water (2 mL) and stirred at room temperature for 10 minutes. This solution (1 mL) was added dropwise to a mixture of crude compound 6 (51.0 mg), sodium L-(+)-ascorbate (88.4 mg, 0.46 mmol), copper sulfate (114 mg, 0.46 mmol), tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (69.2 mg, 0.13 mmol), t-butanol (3 mL), purified water (3 mL), and acetonitrile (1.5 mL). The mixture was protected from light and stirred at room temperature for 18 hours. The solution was diluted with purified water / acetonitrile (9:1) and purified by reverse-phase column chromatography (Gradient: 10% for 8 min; 10–80% for 40 min; 80–100% for 10 min; 100% for 10 min CHCN containing 100 mM TEAA in H2O containing 100 mM TEAA, MPLC (Sfaer C18 D-Duo 100Å 30 μm), flow rate 12 mL / min) followed by HPLC (Gradient: 30–100% for 50 min; 100% for 30 min 100 mM TEAA in H2O containing 100 mM TEAA, ODS-3 column (GL Sciences Inc.) 5 mL / min). This was followed by further purification by reverse-phase column chromatography (Gradient: 1% for 7 min; 1-100% for 1.5 min, 100% for 4.2 min CHOH in HO, MPLC (Sfaer C18 D-Duo 100Å 30 μm), flow rate 12 mL / min) to give compound 7 (3.9 mg, 1.9 μmol, 5 steps 1.7%). 1H NMR (500 MHz, MeOD): δ 9.68 (m, 1H), 9.63 (m, 5H), 9.51 (d, J= 8.2 Hz, 1H), 9.12 (d, J = 2.1Hz, 1H), 8.36 (m, 6H), 8.14 (s,1H), 7.93 (dd, J = 2.3, 8.1 Hz, 1H), 4.86, (t, J = 6.3 Hz, 2H), 4.39, (d, J = 7.2 Hz, 2H), 3.42, (d, J = 6.2 Hz, 2H), 3.04, (dd, J = 7.5,7.99 Hz, 24H), 2.63 (m, 24H), 2.23 (2H, t, J = 7.2 Hz, 2H), 1.89 (m, 6H), 1.57 (m, 14H), -1.05 (m, 4H), -2.28 (m, 4H), -2.92 (s, 12H)

[0238]

[0239] (8) Synthesis of Compound 8 Compound 7 (4.5 mg, 2.2 μmol), di(succinimidyl)carbonate (12.7 mg, 0.050 mmol), triethylamine (6.1 mg), and dimethyl sulfoxide (500 μL) were mixed and incubated at room temperature for 18 hours in the dark. 15 mL of diethyl ether was added to the solution, and the mixture was centrifuged and the supernatant was removed. This process was repeated three times, then the mixture was air-dried and dissolved in 300 μL of dimethyl sulfoxide to obtain a dimethyl sulfoxide solution of compound 8 (2.4 mM).

[0240]

[0241] Synthesis Example 2 Synthesis of Compound 15 (1) Synthesis of Compound 9 1,3-Diiminoisoindoline (1.47 g, 10.1 mmol) was dissolved in quinoline (15 ml) and stirred at room temperature for 10 minutes under an argon atmosphere. Tetrachlorosilane (1 ml) was added dropwise to this solution, and the mixture was stirred at 200°C for 2 hours. After cooling to room temperature and diluting with purified water (100 mL), the precipitate was collected by suction filtration. This was washed with purified water and methanol to obtain crude compound 9.

[0242]

[0243] (2) Synthesis of Compound 10 The entire amount of Compound 9 obtained in (1) above was dissolved in 2 M NaOH aq. / EtOH (1:1) (60 mL) and stirred at 70°C for 2 hours. After cooling to room temperature and diluting with water, the precipitate was collected by suction filtration. This was washed with purified water and methanol to obtain crude Compound 10.

[0244]

[0245] (3) Synthesis of Compound 11: The entire amount of compound 10 obtained in (2) above was dissolved in dehydrated pyridine (40 mL). 3-aminopropyldimethylethoxysilane (2 mL) was added dropwise under stirring under an argon atmosphere. The mixture was shielded from light and stirred at 110 °C for 18 hours. The solution was concentrated using a rotary evaporator and then purified by column chromatography (Gradient: 0% for 9 min; 0-1% for 12 min; 1% for 7 min; 1-5% for 15 min; 5% for 7 min, 5-10% for 8 min; 10% for 8 min CHOH in CHCl; MPLC (CHROMATOREX Q-PACK NH 60 SIZE60) (60 μm, 27 × 100 mm), flow rate 20 mL / min) to obtain compound 11 (260 mg, 0.32 mmol, 3 steps 3.2%). 1 H NMR (500 MHz, CDCl3): δ 9.66 (m, 8H), 8.34 (m, 8H), 1.18 (t, J = 7.2 Hz 4H), 0.82 (brs, 4H), -1.22 (m, 4H), -2.29 (m, 4H), -2.85 (m, 12H) 13 C NMR (126 MHz, CDCl3); δ 144.8, 136.2, 131.0, 123.7, 44.4, 26.2, 12.6, -3.2,

[0246]

[0247] (4) Synthesis of Compound 12: Compound 11 (22.4 mg, 0.012 mmol), diisopropylethylamine (1.8 mL), 1-hydroxybenzotriazole (237 mg, 1.75 mmol), and N,N-diisopropylcarbodiimide (WSC-HCl, 99.0 mg, 0.52 mol) were added to dichloromethane (240 mL) and stirred at room temperature in the dark. A solution of 11-azido-3,6,9-trioxaundecanoic acid (43.4 mg, 0.186 mmol) in dimethylformamide (24 mL) was added dropwise to the solution and stirred at room temperature for 48 hours. After concentrating the mixture on an evaporator, the mixture was dissolved in dichloromethane (100 mL) and washed three times with purified water (50 mL). The organic layer was dried over sodium sulfate and purified by column chromatography (gradient: 1% for 5 min; 1-5% for 20 min; 20% for 12 min; 20-100% for 15 min CHOH in CHCl, MPLC (Yamazen Corporation Universal PLC)). TM Crude compound 12 (40.0 mg) was obtained using a column (Premium Amino) (30 μm, 2.3 × 12.3 cm, flow rate 10 ml / min).

[0248]

[0249] (5) Synthesis of Compound 13. Crudely purified compound 12 (38.8 mg) was dissolved in anhydrous methanol (3 mL), diisopropylethylamine (1.0 mL), and 1,3-propane sultone (0.5 mL). After freeze-degassing, the solution was stirred at 50 °C for 18 hours under an argon atmosphere, shielded from light. After concentration using a rotary evaporator, the solution was diluted with a mixture of purified water and acetonitrile (9:1) and crudely purified by reverse-phase column chromatography (gradient: 20% for 5 min; 20-80% for 40 min; 80-100% for 5 min; 100% for 10 min; CHCN containing 100 mM TEAA in HO containing 100 mM TEAA, MPLC (Sfaer C18 D-Duo 100Å 30 μm 12 g)). Compound 13 (41.6 mg, 0.026 mmol, 2 steps, 14.2%) was obtained by reverse-phase column chromatography (Gradient: 1% for 7 min; 1-100% for 1.5 min, 100% for 4.2 min CHOH in HO, MPLC (Sfaer C18 D-Duo 100 Å 30 μm), flow rate 12 ml / min). 1 H NMR (500 MHz, MeOD, KOD): δ 9.65 (m, 8H), 8.36 (m, 8H), 3.62-3.34 (m, 12H), 3.27-2.93 (m, 18H), 3.63 (t, J = 5.2Hz, 2H), 2.84-2.53 (m , 14H), 1.91 (t, J = 8.9 Hz, 2H), 1.61 (m, 8H), 1.04 (brs, 5H) , -1.13 (m, 4H), -2.24 (m,2H), -2.30 (m, 2H), -2.89 (s, 6H), -2.95 (s, 6H) 13C NMR (126 MHz, MeOD): δ 170.3, 148.7, 135.7, 136.0, 131.5, 123.5, 123.4, 70.5, 70.0, 70.0, 69.6, 69.4, 58.76, 50.31, 46.4,00. 40.3, 13.7, 12.4, 12.3, 11.15, 7.13, -4 -4.7

[0250]

[0251] (6) Synthesis of Compound 14: 5-Hexynoic acid (91.8 mg, 0.80 mmol) and sodium bicarbonate (218 mg, 2.59 mmol) were dissolved in purified water (10 mL) and stirred at room temperature for 10 minutes. This solution (1 mL) was added dropwise to a mixture of compound 13 (22.4 mg, 0.012 mmol), sodium L-(+)-ascorbate (36.5 mg, 0.18 mmol), copper sulfate (23.0 mg, 0.092 mmol), and tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (8.97 mg, 0.017 mmol) in t-butanol (3 mL), purified water (3 mL), and acetonitrile (1.5 mL). The mixture was protected from light and stirred at room temperature for 18 hours. This was diluted with a mixture of purified water and acetonitrile (9:1) and crudely purified by reverse-phase column chromatography (Gradient: 10% for 8 min; 10-80% for 40 min; 80-100% for 10 min; 100% for 10 min; CH3CN containing 100 mM TEAA in HO containing 100 mM TEAA, MPLC (Sfaer C18 D-Duo 100Å 30 μm 12 g), flow rate 12 mL / min). Further purification by HPLC (Gradient: 30-100% for 50 min; 100% for 30 min, CHCN containing 100 mM TEAA in HO containing 100 mM TEAA, ODS-3 column (GL Sciences Inc.) 5 mL / min) and reverse-phase column chromatography (Gradient: 1% for 7 min; 1-100% for 1.5 min, 100% for 4.2 min, CHOH in HO, MPLC (Sfaer C18 D-Duo 100 Å 30 μm), flow rate 12 mL / min) gave compound 14 (3.5 mg, 2.1 μmol, 29%). 1H NMR (500 MHz, CDCl3) : δ 9.64 (m, 8H), 8.38 (m, 8H), 7.58 (s, 1H), 4.33 (t, J = 5.2 Hz, 2H), 3.63 (t. J = 5.2 Hz, 2H), 3.58 (s, 2H), 3.45-3.32 (m, 8H), 2.68 (m, 12H), 2.57 (t, J = 7.6 Hz, 2H), 2.11 (t, J = 7.6 Hz, 2H), 1.80 (m, 2H), 1.91 (t, 2H), 1.62 (m, 8H), -1.19 (m, 4H), -2.23 (t, J = 8.2 Hz, 2H), -2.32 (m, 2H), -2.90 (s, 6H), -2.96 (s, 6H)

[0252]

[0253] (7) Synthesis of Compound 15. Compound 14 (1.5 mg, 0.88 μmol), di(succinimidyl)carbonate (3.5 mg, 0.014 mmol), and triethylamine (1.4 mg) were dissolved in dimethyl sulfoxide (500 μL) and incubated at room temperature for 18 hours in the dark. 15 mL of diethyl ether was added to the solution, and the mixture was centrifuged and the supernatant was removed. This process was repeated three times, followed by air drying and dissolving in 300 μL of dimethyl sulfoxide to obtain a dimethyl sulfoxide solution of compound 15 (5.5 mM).

[0254]

[0255] Synthesis Example 3: Synthesis of Compound 8-cet and Compound 15-cet. The anti-EGFR antibody (cetuximab) contained in 100 mg (0.5 mL) of Erbitux injection was applied to a PD MiniTrap G-25 and recovered with 1 mL of PBS. (Solution 1) The concentration of Solution 1 was calculated from the absorbance (280 nm) based on the concentration of Erbitux injection (5 mg / mL). Cetuximab (100 μg), Compound 8 (prepared in Synthesis Example 1) or Compound 15 (5 nmol) (prepared in Synthesis Example 2), and PBS (100 μL) were incubated at room temperature for 1.5 hours in the dark. The mixture was then purified by size exclusion chromatography and concentrated by ultrafiltration to obtain Compound 8-labeled cetuximab (Compound 8-cet) and Compound 15-labeled cetuximab (Compound 15-cet). The antibody concentration of the compound-labeled cetuximab was determined using a BCA assay (Pierce BCA Protein Assay Kit). The concentrations of antibody-labeled Compound 8 or Compound 15 were calculated from the molar extinction coefficients measured at 681 nm or 678 nm. The resulting drug-antibody ratios (DARs) of Compound 8-cet and Compound 15-cet per antibody molecule were estimated to be 1.4 and 1.5, respectively.

[0256] Synthesis Example 4: Synthesis of Compound 8-basiliximab. The anti-IL2RA antibody (basiliximab) contained in Simulect Intravenous Injection 20 mg (0.5 mL) was applied to a PD MiniTrap G-25 and recovered with 1 mL of PBS. The antibody concentration of the recovered solution was calculated from the absorbance at 280 nm based on the Simulect Intravenous Injection concentration (2.2 mg / mL). Basiliximab (100 μg) and Compound 8 (10 nmol) were mixed in PBS (100 μL) and incubated at room temperature for 1.5 hours in the dark. Compound 8-labeled basiliximab (compound 8-bas, also referred to as compound 8-bas) was obtained by further purification by size exclusion chromatography and concentration by ultrafiltration. The antibody concentration of the compound-labeled basiliximab was determined by BCA assay (Pierce BCA Protein Assay Kit). The concentration of antibody-labeled compound 8 was calculated from the molar extinction coefficient by measuring the absorbance at 681 nm. The drug-antibody ratio (DAR) of the resulting compound 8-basiliximab, i.e., the number of labeled molecules per antibody molecule, was estimated to be 2.3.

[0257] Example 1 Photoreactivity of Compound 7 and Compound 14 A PBS solution of 1 μM Compound 7 or Compound 14 in the presence of 5 mM cysteine ​​was irradiated with an LED light source (L660-66-60; Marubeni America Co., Santa Clara, CA) at 660 nm wavelength and 30 mW, and the absorbance and fluorescence intensity (e.g., at 630 nm) were measured every 10 minutes.

[0258] Figure 1(A) is a graph showing the absorption of compound 7 at 681 nm. The vertical axis represents absorption, and the horizontal axis represents irradiation time. Figure 1(B) is a graph showing the absorption of compound 14 at 678 nm. The vertical axis represents absorption, and the horizontal axis represents irradiation time. Figure 1(C) is a graph showing the fluorescence intensity of compound 7 at 695 nm. The vertical axis represents fluorescence intensity, and the horizontal axis represents irradiation time. Figure 1(D) is a graph showing the fluorescence intensity of compound 14 at 687 nm. The vertical axis represents fluorescence intensity, and the horizontal axis represents irradiation time. For both compounds 7 and 14, near-infrared light absorption and attenuation of absorption and fluorescence depending on the irradiation time of near-infrared light were confirmed.

[0259] Example 2 Induction of Cell Death by Photoimmunotherapy Probes Using the probes of compound 8-cet and compound 15-cet prepared in Synthesis Example 3, cell death in Difi cells was examined.

[0260] (1) Cytotoxicity Test: DiFi-BRAF cells, which were created by forcibly expressing an active BRAF mutant gene in the EGFR-expressing colon cancer cell line DiFi, were seeded at 10,000 cells (100 μL) in a 96-well plate and incubated at 37°C for 24 hours. The medium was then removed, and 100 μL of a medium solution containing compound 8 at concentrations of 0, 0.2, 2, or 20 μg / mL was added to each well. The cells were then incubated at 37°C for 1 hour. This process of removing the medium and adding 100 μL of phenol red-free medium was repeated twice. Each well was illuminated at 100 mW / cm using a 690 nm LED light source (L690-66-60; Marubeni America Co., Santa Clara, CA). 2 The cells were irradiated with light for 80 seconds and incubated at 37°C for 24 hours. WST-8 reagent (10 μL) was added to the cells, and the cells were incubated at 37°C for 1.5 hours. The absorbance (450 nm) was measured to determine the cell viability (Figure 2).

[0261] As shown in Figure 2, the dye alone did not affect cell viability even with increasing dye concentration.

[0262] (2) Concentration-dependent cytotoxicity test of antibody-dye conjugates DiFi-BRAF cells were seeded at 10,000 cells (100 μL) in a 96-well plate and incubated at 37°C for 24 hours. The medium was then removed, and 100 μL of a medium solution containing compound 8-cet or compound 15-cet at antibody concentrations of 0, 0.5, 1, 5, 10, or 15 μg / mL was added, followed by incubation at 37°C for 1 hour. This process of removing the medium and adding 100 μL of phenol red-free medium was repeated twice. A 690 nm laser light source was used to illuminate each well at 100 mW / cm. 2The cells were irradiated with light for 160 seconds and incubated at 37°C for 24 hours. WST-8 reagent (10 μL) was added to the cells, and the cells were incubated at 37°C for 1.5 hours. The absorbance (450 nm) was measured to determine the cell viability (Figure 3).

[0263] As shown in Figure 3, for both compound 8-cet and compound 15-cet, cell viability decreased with increasing concentration of the antibody-dye conjugate.

[0264] (3) Light Energy-Dependent Cytotoxicity Test: DiFi-BRAF cells were seeded at 10,000 cells (100 μL) in a 96-well plate and incubated at 37°C for 24 hours. The medium was then removed, and 100 μL of a medium solution containing compound 8-cet or compound 15-cet at antibody concentrations of 0, 0.2, 2, or 20 μg / mL was added, followed by incubation at 37°C for 1 hour. This process of removing the medium and adding 100 μL of phenol red-free medium was repeated twice. A laser light source with a wavelength of 690 nm was used to illuminate each well at 100 mW / cm. 2 The cells were irradiated with light for 0, 10, 20, 40, 80, 160, and 320 seconds and then incubated at 37°C for 24 hours. WST-8 reagent (10 μL) was added to the cells, and the cells were incubated at 37°C for 1.5 hours. The absorbance (450 nm) was measured to determine the cell viability (Figure 4).

[0265] As shown in FIG. 4, in both the case of compound 8-cet and compound 15-cet, the cell viability decreased as the irradiation energy increased.

[0266] Thus, we synthesized compound 8, a water-soluble phthalocyanine silicon complex, an IR700 analogue with a meta-substituted linker. We then synthesized a probe conjugated to the anti-EGFR (epidermal growth factor receptor) antibody cetuximab. This probe was added to the culture medium of EGFR-expressing cells, and cell viability was measured after irradiation with 690 nm light. The probe demonstrated photoimmunoactivity comparable to that observed in studies using IR700 (see, for example, Molecular Oncology 2014, 3, 620-632, DOI:10.1016 / j.molonc.2014.01.006 and Synlett 2021, 32, 1098-1103, DOI:10.1055 / a-1503-6425).

[0267] Example 3 Evaluation of reactivity of compound 7 to near-infrared light Compound 7 (final concentration 1 μM) and cysteine ​​(final concentration 5 mM) were dissolved in PBS and exposed to 100 mW / cm 2 was irradiated for 160 seconds (light intensity 16 J / cm 2 ) When the solution was analyzed by HPLC before and after light irradiation, the peak of compound 7 disappeared upon light irradiation, and new peaks a and b, which are presumably due to the elimination of the water-soluble axial ligand, were generated, demonstrating that compound 7 is reactive to near-infrared light (Figure 5).

[0268] Example 4 Morphological Changes in Target Cells by Near-Infrared Light Irradiation DiFi-BRAF cells were seeded at 10,000 cells (200 μL) on a glass-bottom dish and incubated at 37°C for 24 hours. Compound 8-cetuximab was then added to a concentration of 1 μg / mL, and the cells were incubated at 37°C for 1 hour. This procedure of removing 200 μL of medium and adding 200 μL of new medium was repeated twice, and the cells were then exposed to near-infrared light at a wavelength of 650 nm (1 mW / cm 2 ) for 1000 seconds (light intensity 1 J / cm 2Bright-field images of cells were taken using an FV3000 (Olympus) before irradiation (Pre), immediately after irradiation (0 min), 5 min, 15 min, and 30 min after irradiation. Cell morphological changes were observed immediately after near-infrared light irradiation (Figure 6). Similar bleb formation has also been reported as a characteristic cell morphological change after near-infrared light irradiation in photoimmunotherapy using an IR-700 antibody conjugate (ACS Cent. Sci. 2018, 4, 1559-1569).

[0269] Example 5: Selective Cytotoxicity of HTLV-1-Infected Cells by Compound 8-bas. HTLV-1-infected cell lines were MT-1 (JCRB Cell Bank Cell Registration No. JCRB1209), MT-2 (JCRB Cell Bank Cell Registration No. JCRB1210), MT-4 (JCRB Cell Bank Cell Registration No. JCRB0135), and ATN-1 (RIKEN BRC Resource Cell No. RCB1440). HTLV-1-uninfected cell lines were MOLT-4 (JCRB Cell Bank Cell Registration No. JCRB9031) and TALL-1 (RIKEN BRC Resource Cell No. RCB2084). 10,000 cells of each type were seeded into a 96-well plate and incubated at 37°C for 24 hours. Then, 50 μL of medium was removed, and 50 μL of a medium solution containing compound 8-basiliximab at a concentration of 20 μg / mL was added. The cells were then incubated at 37°C for 1 hour. After centrifugation at 300 × g for 3 minutes, 80 μL of medium was removed and 80 μL of phenol red-free medium was added. This procedure was repeated six times. Each well was illuminated with a 690 nm LED light source at 100 mW / cm. 2 The samples were irradiated with light of 32 J / cm for 320 seconds and then incubated at 37°C for 24 hours (light intensity 32 J / cm 2 WST-8 reagent (20 μL) was added to the mixture, which was then incubated at 37°C for 1.5 hours. The absorbance at a wavelength of 450 nm was measured to determine the cell viability (FIG. 7).

[0270] In the HTLV-1-infected cell group to which compound 8-basiliximab was added, cell viability was significantly reduced compared to the groups to which compound 8 alone or basiliximab alone was added.

[0271] Example 6: Near-infrared light-dependent cytotoxicity by compound 8-basiliximab against a mixed cell system infected with and uninfected by HTLV-1. The HTLV-1-infected cell line, MT-1 (10,000 cells), was suspended in PBS (1 mL) and centrifuged at 300 × g for 3 minutes. The supernatant was removed and then suspended in PBS (100 μL). Calcein-AM (4 μL) was added and incubated at room temperature for 30 minutes. The cells were then suspended in PBS (1 mL), centrifuged at 300 × g for 3 minutes, and the supernatant was removed. After repeating this procedure twice, calcein-labeled MT-1 (10,000 cells), unlabeled HTLV-1-uninfected cell line, MOLT-4 (10,000 cells), and compound 8-bas (20 μg) were mixed in medium (200 μL) and incubated for 1 hour. The mixture was centrifuged at 300 × g for 3 minutes, after which 200 μL of medium was removed and 200 μL of phenol red-free medium was added. This procedure was repeated twice, and the cells were suspended in 2 mL of phenol red-free medium. 1 mL of each was then seeded into a 12-well plate. An LED light source with a wavelength of 690 nm was used, and the illumination was 100 mW / cm. 2 After irradiating the well plate with light for 320 seconds, the well plate was incubated at 37°C for 24 hours (light intensity 32 J / cm 2The mixture was centrifuged at 300 × g for 3 minutes, the supernatant was removed, and 200 μL of 4% paraformaldehyde phosphate buffer was added and incubated at room temperature for 15 minutes. The mixture was centrifuged at 200 × g for 5 minutes at room temperature, and the supernatant was removed. The pellet was then suspended in 200 μL of PBS containing 1% BSA, slowly pipetted using a wide-mouth 1 ml tip, and centrifuged at 200 × g for 5 minutes at room temperature, and the supernatant was removed. The precipitate was then suspended in 600 μL of PBS containing 1% BSA, slowly pipetted using a wide-mouth 1 ml tip, and used as the sample for FACS analysis. A control sample not exposed to near-infrared light was prepared in the same manner. The results of flow cytometry analysis are shown in Figure 8. The horizontal axis (Alexa Fluor 488 channel) shows calcein-derived fluorescence (maximum fluorescence wavelength 515 nm), and the vertical axis (Alexa Fluor 700 channel) shows compound 8-derived fluorescence (maximum fluorescence wavelength 681 nm). In the results without near-infrared light irradiation (left), the cells were separated into upper right and lower left groups, indicating that compound 8-basiliximab selectively bound to calcein-labeled MT-1 cells. With near-infrared light irradiation (right), it can be seen that only the calcein-labeled MT-1 (HTLV-1-infected cell line) in the upper right was selectively damaged.

[0272] Example 7: Near-infrared light-dependent cytotoxicity of compound 8-basiliximab on cells derived from an ATL patient. Compound 8-basiliximab (20 μg) was added to 20,000 cells derived from the blood of an ATL patient and incubated in medium (200 μL) for 1 hour. Five mL of phenol red-free medium was added, and the cells were centrifuged at 300 × g for 3 minutes. After that, 5 mL of medium was removed, and 5 mL of phenol red-free medium was added. The cells were then centrifuged at 300 × g for 3 minutes, 5 mL of medium was removed, and 2 mL of phenol red-free medium was added. The resulting cell suspension was seeded in 1 mL aliquots into a 12-well plate. An LED light source with a wavelength of 690 nm was used, and the illumination was 100 mW / cm. 2 After irradiating the well plate with light for 640 seconds, the well plate was incubated at 37°C for 24 hours (light intensity 64 J / cm 2The cells were then stained with 7-AAD Viability Staining Solution (BioLegend, 420404), APC-labeled anti-CD3 antibody (BioLegend, 317318), APC-Cy7-labeled anti-CD4 antibody (BioLegend, 300518), and FITC-labeled anti-human IgG antibody (Abcam, AB6854), and analyzed by flow cytometry. (7-AAD was used as a marker to exclude dead cells. The CD3-positive fraction of live cells was narrowed down to identify T cells. The human IgG antibody was used to bind to basiliximab, which binds to IL2RA (also known as CD25). The CD4-positive and IL2RA-positive fraction of CD3-positive T cells was determined to be HTLV-1-infected.) The results of flow cytometry are shown in Figure 9. The horizontal axis (APC-Cy7 channel) shows the fluorescence derived from CD4, and the vertical axis (FITC channel) shows the fluorescence derived from IL2RA (also known as CD25) bound to human IgG (basiliximab). Near-infrared light irradiation selectively reduced the IL2RA-positive fraction with FITC signals, suggesting that HTLV-1-infected cells were selectively damaged (Figure 9).

Claims

1. A phthalocyanine dye represented by formula (I): where M is selected from Si, Ge, Sn, or Al; L is an optionally substituted triazolyldiyl group or a linker comprising a fused ring of an optionally substituted diazine and a cycloalkane; Q is a reactive group for attachment of the dye to a targeting molecule; R 2 , R 3 , R 7 , and R 8 are each independently selected from optionally substituted alkyl and optionally substituted aryl; R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 are each independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkanoyl, optionally substituted alkoxycarbonyl, optionally substituted alkylcarbamoyl, and a chelating ligand, where R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 At least one of R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 and R 23 are each independently selected from hydrogen, halogen, optionally substituted alkylthio, and optionally substituted alkylamino; and X 1 and X 2 each independently represents a C-C 10 It is alkylene.

2. The phthalocyanine dye according to claim 1, wherein L is a group represented by the following formula (II): wherein Y is selected from a direct bond, oxygen, sulfur, or an optionally substituted nitrogen; 3 and X 4 each independently represents a C-C 10 and Z is an optionally substituted triazolyldiyl group or a fused ring of an optionally substituted diazine and a cycloalkane.

3. The phthalocyanine dye of claim 1, wherein Q is selected from an amine reactive chemical group, a sulfhydryl reactive chemical group, an activated ester, an acyl halide, an alkyl halide, an anhydride group, a carboxy, a carboxylate, a carboxylate, a carbodiimide, a carbonate, a carbamate, a haloacetamide, an isocyanate, an isothiocyanate, a maleimide, an N-hydroxysuccinimide ester, a phosphoramidite, a platinum complex, a sulfonate ester, and a thiocyanate.

4. A pharmaceutical agent comprising the phthalocyanine dye of any one of claims 1 to 3 conjugated to a targeting molecule that binds to an antigen, protein, or cell surface target molecule.

5. The agent of claim 4, wherein the targeting molecule is a protein, peptide, or small molecule.

6. The agent of claim 4, wherein the targeting molecule is an antibody or an antibody fragment.

7. The agent according to claim 4 for inducing cytotoxicity by apoptosis in target cells.

8. The drug according to claim 7, wherein the induction of cytotoxicity is brought about by administering the drug to the tumor cells and irradiating the tumor cells with light after administering the drug.

9. The drug according to claim 4 for treating a tumor in a subject.

10. A method for producing a phthalocyanine dye represented by formula (IV), comprising reacting a compound represented by formula (III) with a reagent (B) to obtain a compound represented by formula (IV). In the formulas (III) and (IV), M is selected from Si, Ge, Sn, or Al; R 2 , R 3 , R 7 , and R 8 are each independently selected from optionally substituted alkyl and optionally substituted aryl; R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 are each independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkanoyl, optionally substituted alkoxycarbonyl, optionally substituted alkylcarbamoyl, and a chelating ligand, where R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 At least one of R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 and R 23 are each independently selected from hydrogen, halogen, optionally substituted alkylthio, and optionally substituted alkylamino; Y is selected from a direct bond, oxygen, sulfur, or optionally substituted nitrogen; X 1 and X 2 each independently represents a C-C 10 is alkylene, and X 3 and X 4 each independently represents a C-C 10 is selected from alkylene, polyethylene glycol (PEG), phenylene, ester, amide, carbamate, or a combination of two or more thereof; Z is an optionally substituted triazolyldiyl group or a fused ring of an optionally substituted diazine and a cycloalkane; P 1 is selected from among an amine-reactive chemical group, a sulfhydryl-reactive chemical group, an activated ester, an acyl halide, an alkyl halide, an acid anhydride group, a carboxy, a carboxylate, a carboxylate, a carbodiimide, a carbonate, a carbamate, a haloacetamide, an isocyanate, an isothiocyanate, a maleimide, an N-hydroxysuccinimide ester, a phosphoramidite, a platinum complex, a sulfonate ester, and a thiocyanate; A has a first functional group; and reagent (B) has a second functional group that reacts with the first functional group of A by a Click reaction.

11. A method for producing a phthalocyanine dye represented by formula (VII), comprising reacting a compound represented by formula (VI) with an azide (X) to obtain a compound represented by formula (VII). In formulas (VI), (VII), and (X), M is selected from Si, Ge, Sn, or Al; R 2 , R 3 , R 7 , and R 8 are each independently selected from optionally substituted alkyl and optionally substituted aryl; R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 are each independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkanoyl, optionally substituted alkoxycarbonyl, optionally substituted alkylcarbamoyl, and a chelating ligand, where R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 At least one of R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 and R 23 are each independently selected from hydrogen, halogen, optionally substituted alkylthio, and optionally substituted alkylamino; Y is selected from a direct bond, oxygen, sulfur, or optionally substituted nitrogen; X 1 and X 2 each independently represents a C-C 10 is alkylene, and X 3 and X 4 each independently represents a C-C 10 is selected from alkylene, polyethylene glycol (PEG), phenylene, ester, amide, carbamate, or a combination of two or more thereof; Z is an optionally substituted triazolyldiyl group; P 1 is selected from among amine reactive chemical groups, sulfhydryl reactive chemical groups, activated esters, acyl halides, alkyl halides, acid anhydride groups, carboxy, carboxylate, carboxylate, carbodiimide, carbonate, carbamate, haloacetamide, isocyanate, isothiocyanate, maleimide, N-hydroxysuccinimide ester, phosphoramidite, platinum complex, sulfonate ester, and thiocyanate.

12. A method for producing a phthalocyanine dye represented by formula (I), comprising producing a compound represented by formula (IV) by the method described in item 10, and adding P of formula (IV) to the compound represented by formula (IV) produced. 1 or preparing a compound represented by formula (VII) by the method according to claim 11, and reacting the compound represented by formula (VII) with a compound having a reactive functional group to obtain a compound represented by formula (I): 1 reacting a group with a compound having a reactive functional group to obtain a compound represented by formula (I): (In formula (I), M is selected from Si, Ge, Sn, or Al, L is a group represented by the following formula (II), -X 4 -ZX 3 -Y- Formula (II) (wherein, Y is selected from a direct bond, oxygen, sulfur, or optionally substituted nitrogen; X 3 and X 4 each independently represents a C-C 10 R is an alkylene, polyethylene glycol (PEG), phenylene, ester, amide, carbamate, or a combination of two or more thereof; Z is an optionally substituted triazolyldiyl group or an optionally substituted fused ring of a diazine and a cycloalkane; Q is a reactive group for attachment of the dye to a targeting molecule; R 2 , R 3 , R 7 , and R 8 are each independently selected from optionally substituted alkyl and optionally substituted aryl; R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 are each independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkanoyl, optionally substituted alkoxycarbonyl, optionally substituted alkylcarbamoyl, and a chelating ligand, where R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 At least one of R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 and R 23 are each independently selected from hydrogen, halogen, optionally substituted alkylthio, and optionally substituted alkylamino; and X 1 and X 2 each independently represents a C-C 10 It is alkylene.

13. Any of the following compounds (iii) to (vi): (In formula (iii), M is selected from Si, Ge, Sn, or Al; R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 and R 23 are each independently selected from hydrogen, halogen, optionally substituted alkylthio, and optionally substituted alkylamino; Y is selected from a direct bond, oxygen, sulfur, or optionally substituted nitrogen; and X 3 each independently represents a C-C 10 alkylene.) In formula (iv), M is selected from Si, Ge, Sn, or Al; R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 and R 23 are each independently selected from hydrogen, halogen, optionally substituted alkylthio, and optionally substituted alkylamino; R 24 and R 25 are each independently C1-C 10 an alkyl group; and Y is selected from a direct bond, oxygen, sulfur, or an optionally substituted nitrogen; X 3 each independently represents a C-C 10 alkylene.) (In formula (v), M is selected from Si, Ge, Sn, or Al; R 2 , R 3 , R 7 , and R 8 are each independently selected from optionally substituted alkyl and optionally substituted aryl; R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 and R 23 are each independently selected from hydrogen, halogen, optionally substituted alkylthio, and optionally substituted alkylamino; and Y is selected from a direct bond, oxygen, sulfur, or optionally substituted nitrogen; X 1 and X 2 each independently represents a C-C 10 is alkylene, and X 3 each independently represents a C-C 10 alkylene.) In formula (vi), M is selected from Si, Ge, Sn, or Al; R 2 , R 3 , R 7 , and R 8 are each independently selected from optionally substituted alkyl and optionally substituted aryl; R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 are each independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkanoyl, optionally substituted alkoxycarbonyl, optionally substituted alkylcarbamoyl, and a chelating ligand, where R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 At least one of R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 and R 23 are each independently selected from hydrogen, halogen, optionally substituted alkylthio, and optionally substituted alkylamino; and Y is selected from a direct bond, oxygen, sulfur, or optionally substituted nitrogen; X 1 and X 2 each independently represents a C-C 10 is alkylene, and X 3 each independently represents a C-C 10 alkylene.)

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