Novel prodrug-type anticancer agent targeting cancer-specific enzyme activity

Abstract

[Problem] To provide a novel compound promising as a prodrug-type anticancer agent that exhibits a sufficient anticancer effect and is capable of reducing adverse effects. [Solution] A compound represented by general formula (I) or a pharmaceutically acceptable salt of the compound. Formula (I): Y-T-(X1)n

Description

Novel prodrug-type anticancer drugs targeting cancer-specific enzyme activity

[0001] The present invention relates to novel compounds that are promising as prodrug-type anticancer agents, and to prodrug-type anticancer agents using the compounds.

[0002] SN38 is a compound that exhibits antitumor effects by inhibiting topoisomerase I. Due to its high lipid solubility, SN38 is difficult to administer alone, and therefore, in cancer chemotherapy, it is currently mainly used as irinotecan (CPT-11), a compound with a carboxylesterase (CES) hydrolysis site added to its molecular structure to make it water-soluble (Non-Patent Documents 1 and 2).

[0003] CPT-11 is a prodrug designed to release SN38 upon side chain degradation by CES. However, CES is not a cancer site-specific enzyme and is highly expressed in various organs (especially the colon and liver), making it unable to release SN38 specifically at the cancer site, resulting in organ damage. It has also been reported that CPT-11 itself pharmacologically inhibits acetylcholinesterase. For these reasons, serious side effects have been reported in clinical settings, including acute cholinergic syndrome and delayed severe diarrhea and neutropenia.

[0004] Therefore, if a new small molecule prodrug-type anticancer drug could be developed that targets a cancer-specific enzyme rather than CES and releases SN38, it would be possible to reduce side effects and achieve treatment that is specific to the cancer site, but this has not yet been realized.

[0005] Annals of Oncology 13: 1841-1851, 2002Int. J. Mol. Sci. 2020, 21, 4919

[0006] An object of the present invention is to provide novel compounds that are promising as prodrug-type anticancer agents that exhibit sufficient anticancer activity and can reduce side effects.

[0007] Our laboratory has previously developed a group of fluorescent probes capable of detecting various glycosidase activities, and by using these probes to screen cancerous and normal tissues in human clinical specimens, we have discovered various cancer-cell-specific glycosidase activities. In particular, we have found that fluorescent probes targeting β-galactosidase and β-hexosaminidase are effective in specifically illuminating lung cancer, and have newly discovered that these target enzymes, β-galactosidase 1 (GLB1), β-hexosaminidase A, and β-hexosaminidase B, are highly expressed in lung cancer tissue.

[0008] Based on these research results from our laboratory, the present inventors conceived the idea that by introducing a substrate moiety that targets the cancer cell-specific enzyme activity discovered by screening of fluorescent probes into the molecules of anticancer drugs such as SN38, it might be possible to develop new prodrug-type anticancer drugs based on anticancer drugs such as SN38 that would selectively show efficacy at cancer sites and have reduced side effects (see Figure 1).As a result of extensive research, they have completed the present invention.

[0009] That is, the present invention provides: [1] a compound represented by the following general formula (I) or a pharmaceutically acceptable salt thereof: Y-T-(X １ ) ｎ (I) (Wherein, Y is -OL １ (1) or -NH-L ２ (2) is selected from L １ is a partial structure of a saccharide, where the partial structure of a saccharide is a structure obtained by removing one hydroxyl group from a saccharide, and L ２ is a monovalent substituent containing an amino acid residue or an oligopeptide residue; T is a linker or bond; X １ is at least one selected from the group consisting of: (1) a binding residue of a biologically active compound having one or more functional groups selected from the group consisting of an aliphatic hydroxyl group, an aromatic hydroxyl group, an amino group, and a carboxyl group; (2) an antibody; and (3) a ligand selected from a small molecule, a peptide, an aptamer, or a hormone-drug conjugate, with the proviso that X １at least one of the following is a binding residue of the biologically active compound; the biologically active compound is an anticancer drug; and n is an integer of 1 or more. [2] The compound according to [1], wherein Y is selected from any of the following groups, or a pharmaceutically acceptable salt thereof. [3] The compound or pharmaceutically acceptable salt thereof according to [1] or [2], wherein the linker is selected from the group consisting of the following structures (a) to (f) and combinations of two or more thereof: (In the formula, R １ ~R ４ may be the same or different, and each independently represents hydrogen, a methyl group, a methoxy group, —COOMe, —NHMe, F, Cl, Br, or NO ２ wherein Me represents a methyl group; R ５ is hydrogen or an alkyl group having 1 to 3 carbon atoms; R ６ is independently in each occurrence hydrogen or an alkyl group having 1 to 3 carbon atoms; provided that: (i) R １ ~R ４ At least one of may be a linking group selected from: Here, R １ ~R ４ When at least one of is a linking group, the linker can be connected to X １ (ii) R １ ~R ４ At least one of may be a linker with a binding site that can be used as a point of attachment to an antibody or ligand: * represents X １ However, when T has a structure that is a combination of two or more of the formulas (a) to (f), X １ * for one or more linkers other than the linker bonded to X may represent the direction of bonding to the linker; １When at least one of the above is an antibody or a ligand, the linkers of formulae (a) to (f) and the linking groups of a1 to a3 can be attached to a linker having a binding site that can be used as a point of attachment to the antibody or ligand in the direction indicated by *.) [4] The compound or pharmaceutically acceptable salt thereof according to any one of [1] to [3], wherein the biologically active compound is selected from any of the following: [5] The compound according to any one of [1] to [4], or a pharmaceutically acceptable salt thereof, wherein the biologically active compound is SN38, topotecan, exatecan, etoposide, doxorubicin, epirubicin, melphalan, MMAE, MMAF, paclitaxel, docetaxel, or cabazitaxel. [6] The compound according to [4], or a pharmaceutically acceptable salt thereof, wherein the biologically active compound is SN38, topotecan, exatecan, etoposide, doxorubicin, epirubicin, melphalan, MMAE, MMAF, paclitaxel, docetaxel, or cabazitaxel. [7] A prodrug-type anticancer agent comprising the compound according to any one of [1] to [6], or a pharmaceutically acceptable salt thereof. [8] A prodrug-type anticancer drug that acts cell-selectively by cancer cell-specific enzymatic activity, comprising the compound according to any one of [1] to [6] or a pharmaceutically acceptable salt thereof. [9] The prodrug-type anticancer drug according to [7] or [8], wherein the enzyme is a peptidase or a glycosidase.

[10] A fluorescent probe for detecting lung cancer, comprising a compound represented by the following general formula (II) or a salt thereof: (In the formula, R １ When present, represent the same or different monovalent substituents present on the benzene ring; R ２ and R ３ each independently represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom; R ４ and R ５ each independently represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom; R ６ represents a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, or a fluorinated alkyl group having 1 to 5 carbon atoms; R ７ and R８ When present, each independently represents an alkyl group or an aryl group having 1 to 6 carbon atoms, and when X is an oxygen atom, R ７ and R ８ is absent; X represents an oxygen atom, a silicon atom, or a carbon atom; n is an integer of 1 to 3; and L is a group of the following formula (1b):

[11] The fluorescent probe according to

[10] , wherein X is an oxygen atom.

[12] R ６ is a fluorinated alkyl group having 1 to 5 carbon atoms.

[13] A companion diagnostic for diagnosing the need for cancer treatment with a prodrug-type anticancer drug comprising a compound represented by the following general formula (I) or a pharmaceutically acceptable salt thereof, the companion diagnostic comprising a compound represented by the following general formula (II) or a salt thereof: Y-T-(X １ ) ｎ (I) wherein Y is selected from any of the following groups: T is a linker or bond: X １ is at least one selected from the group consisting of: (1) a binding residue of a biologically active compound having one or more functional groups selected from the group consisting of an aliphatic hydroxyl group, an aromatic hydroxyl group, an amino group, and a carboxyl group; (2) an antibody; and (3) a ligand selected from a small molecule, a peptide, an aptamer, or a hormone-drug conjugate, with the proviso that X １ at least one of the following is a binding residue of the biologically active compound; the biologically active compound is an anti-cancer drug; and n is an integer of 1 or greater. (In the formula, R １ When present, represent the same or different monovalent substituents present on the benzene ring; R ２ and R ３ each independently represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom; R ４ and R ５ each independently represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom; R ６represents a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, or a fluorinated alkyl group having 1 to 5 carbon atoms; R ７ and R ８ When present, each independently represents an alkyl group or an aryl group having 1 to 6 carbon atoms, and when X is an oxygen atom, R ７ and R ８ is absent; X represents an oxygen atom, a silicon atom, or a carbon atom; n is an integer of 1 to 3; and L is selected from any of the following groups:

[14] A pharmaceutical composition for treating or preventing cancer, comprising the compound according to any one of [1] to [6] or a pharmaceutically acceptable salt thereof.

[15] A pharmaceutical composition for treating or preventing cancer, comprising, in addition to the compound represented by general formula (I) or a pharmaceutically acceptable salt, hydrate, or solvate thereof, another anticancer drug.

[16] The pharmaceutical composition according to

[15] , wherein the other anticancer drug is selected from methotrexate, doxorubicin, cisplatin, or an anti-PD-1 antibody.

[17] A method for treating a cancer patient, comprising the step of administering to the patient a therapeutically effective amount of a prodrug-type anticancer drug according to any one of [7] to [9].

[18] The method according to

[17] , wherein the prodrug-type anticancer drug acts cell-selectively by virtue of an enzymatic activity specific to cancer cells, and the cancer is characterized by a higher expression level of the enzyme.

[19] The method of treatment according to

[17] or

[18] , wherein the prodrug-type anticancer drug acts cell-selectively through enzymatic activity specific to cancer tissue, and the cancer is characterized by a higher expression level of the enzyme.

[20] The method of treatment according to

[18] , wherein the prodrug-type anticancer drug releases anticancer drug molecules by cleavage of the enzyme recognition site and / or linker site due to enzymatic activity specific to cancer cells.

[21] The method of treatment according to

[19] , wherein the prodrug-type anticancer drug releases anticancer drug molecules by cleavage of the enzyme recognition site and / or linker site due to enzymatic activity specific to cancer tissue.

[22] The method of treatment according to any one of

[18] to

[21] , comprising identifying the level of enzymatic activity of the enzyme in a patient's biological sample using one or more enzyme diagnostic agents selected from a visualization agent or an enzyme activity reporter, and defining groups that may respond to treatment as a function of the enzymatic activity.

[23] The method of treatment according to any one of

[18] to

[22] , comprising using the enzyme diagnostic reagent to stratify a set of patients and define a subset of patients who may respond to the prodrug-type anticancer drug.

[24] Use of an enzyme recognition site-linker binding site represented by general formula (III), wherein the enzyme recognition site has an enzymatic activity specific to cancer cells, to activate an antibody or ligand (such as a small molecule, peptide, aptamer, or hormone-drug conjugate) having a payload that is released by cleavage of the binding site by the enzymatic activity specific to the enzyme recognition site-linker binding site: Y-T1 (III) (wherein Y is -OL. １ (1) or -NH-L ２ (2) is selected from L １ is a partial structure of a saccharide, where the partial structure of a saccharide is a structure obtained by removing one hydroxyl group from a saccharide, and L ２ is a monovalent substituent containing an amino acid residue or an oligopeptide residue; T1 is a linker.

[25] Use of an enzyme recognition site-linker binding site represented by general formula (III), wherein the enzyme recognition site has enzymatic activity specific to cancer cells, to prevent activation of a prodrug or binding of an antibody or a ligand until the enzyme recognition site-linker binding site is cleaved by enzymatic activity specific to the binding site.

[26] Use of an enzyme recognition site-linker binding site represented by general formula (III), wherein the enzyme recognition site has enzymatic activity specific to cancer cells, to activate an antibody or a ligand having a payload that is released or activated by cleavage of the enzyme recognition site-linker binding site by enzymatic activity specific to the binding site, in target cells or tissues that overexpress an oncogene.

[27] Use of an enzyme recognition site-linker binding site represented by general formula (III), wherein the enzyme recognition site has an enzymatic activity specific to cancer cells, for targeting cancer.

[0010] The present invention can provide novel compounds that are promising as prodrug-type anticancer agents that exhibit sufficient anticancer activity and can reduce side effects.

[0011] Furthermore, the present invention can provide a novel lung cancer detection probe that is excellent in both sensitivity and specificity, making it possible to provide a highly effective lung cancer detection probe.

[0012]

[0033] Figure 1 shows the concept (a) and specific molecular structure (b) of the SN38-type prodrug developed in the present invention, which exhibits efficacy through cancer cell-specific enzyme activity. Figure 2 shows the structures of 12 types of glycosidase-reactive fluorescent probes used in the screening of glycosidase activity-detecting fluorescent probes in Example 1. Figure 3 shows the results of screening of glycosidase activity-detecting fluorescent probes in human lung cancer specimens. Figure 4 shows the results of a test to identify the target enzyme of HMRef-βGlcNAc. Figure 5 shows the results of fluorescent imaging of human lung cancer using HMRef-βGlcNAc. Figure 6 shows the results of lung cancer imaging (mouse model) following intraperitoneal and systemic administration of HMRef-βGlcNAc. Figure 7 shows the results of a cytotoxicity test using an MTT assay. Figure 8 shows the inhibition of cell proliferation of various cancer cells by SN38-βGal. Figure 9 shows the inhibition of cell proliferation of various cancer cells by SN38-βGlcNAc. Figure 10 shows the inhibition of cell proliferation of various cancer cells by SN38. Figure 11 shows the inhibition of cell proliferation of various cancer cells by CPT-11. 1 shows the results of confirming the reactivity of SN38-βGal with GLB1 by LC-MS analysis. 2 shows the results of pharmacokinetics following intraperitoneal administration (i.p.) of SN38-βGal at 30 mg / kg. 3 shows changes in body weight following repeated administration of the novel prodrug and CPT-11. 4 shows a comparison of liver, kidney, and spleen weights after the test. 5 shows histological images of the small intestine after repeated drug administration (Scale bar = 100 μm). 6 shows histological images of the large intestine after repeated drug administration (Scale bar = 100 μm). 7 shows histological images of the kidney after repeated drug administration (Scale bar = 100 μm). 8 shows histological images of the liver after repeated drug administration (Scale bar = 100 μm). 9 shows histological images of the spleen after repeated drug administration (Scale bar = 100 μm). 10 shows the results of evaluating the AChE inhibitory activity of SN38-βGal, SN38-βGlcNAc, and SN38-αMan. 11 shows the results of evaluating motor activity after drug administration.

[0039] Figure 1 shows the results of therapeutic evaluation of A549 peritoneal dissemination model mice using SN38-βGal.

[0040] Figure 2 shows the results of therapeutic evaluation of A549 lung cancer orthotopic transplant model using SN38-βGal, SN38-βGlcNAc, and CPT-11 (therapeutic experiment 1).

[0041] Figure 3 shows the results of therapeutic evaluation of A549 lung cancer orthotopic transplant model using SN38-βGal and SN38-βGlcNAc (therapeutic experiment 2).

[0042] Figure 4 shows the results of analysis of pharmacokinetic parameters in common marmosets.

[0033] Figure 1 shows the results of a cytotoxicity test using an MTT assay. Figure 2 shows the tumor growth inhibitory effect of SN38-βGlcNAc in H226 subcutaneous tumor model mice. Figure 3 shows the results of a growth inhibitory test in lung squamous cell carcinoma organoids. Figure 4 shows the tumor growth inhibitory effect of SN38-βGlcNAc in PDX model mice. Figure 5 shows the results of a treatment experiment in an orthotopic transplant model of small cell lung cancer. Figure 6 shows an example of fluorescent detection of human small cell lung cancer tissue using HMRef-βGlcNAc. Figure 7 shows the results of a treatment experiment (first time) using SN38-βGal in an SKOV3 ovarian cancer peritoneal dissemination model. Figure 8 shows the results of a treatment experiment (second time) using SN38-βGal in an SKOV3 ovarian cancer peritoneal dissemination model. Figure 9 shows the results of a combined use of an anti-PD-1 antibody and SN38-βGlcNAc. Figure 10 shows the results of a treatment experiment (first time) using KK-SN38 in an SKOV3 ovarian cancer peritoneal dissemination model. 1 shows the results of a second treatment experiment (second time) using KK-SN38 on an SKOV3 ovarian cancer peritoneal dissemination model. Cytotoxicity test results using a JFCR39 cell panel: GI for each cancer cell. ５０The values ​​are shown. Cytotoxicity test results using a JFCR39 cell panel: Showing the cell growth inhibition of each cancer cell line in SN38-αMan. Cytotoxicity test results using a JFCR39 cell panel: Showing the cell growth inhibition of each cancer cell line in KK-SN38. Cytotoxicity test results using a JFCR39 cell panel: Showing the cell growth inhibition of each cancer cell line in QA-SN38. Cytotoxicity test results using a JFCR39 cell panel: Showing the cell growth inhibition of each cancer cell line in TF-SN38. Cytotoxicity test results using a JFCR39 cell panel: Showing the cell growth inhibition of each cancer cell line in NGT-βGlcNAc. Cytotoxicity test results using a JFCR39 cell panel: Showing the cell growth inhibition of each cancer cell line in EXT-βGlcNAc. Cytotoxicity test results using a JFCR39 cell panel: Showing the cell growth inhibition of each cancer cell line in ETP-βGlcNAc. Cytotoxicity test results using a JFCR39 cell panel: Shows the inhibition of cell proliferation of each cancer cell line by DOX-βGlcNAc. Cytotoxicity test results using a JFCR39 cell panel: Shows the inhibition of cell proliferation of each cancer cell line by EPI-βGlcNAc. Cytotoxicity test results using a JFCR39 cell panel: Shows the inhibition of cell proliferation of each cancer cell line by Melphalan-βGlcNAc. Cytotoxicity test results using a JFCR39 cell panel: Shows the inhibition of cell proliferation of each cancer cell line by MMAE-βGlcNAc. Cytotoxicity test results using a JFCR39 cell panel: Shows the inhibition of cell proliferation of each cancer cell line by MMAF-βGlcNAc. Cytotoxicity test results using a JFCR39 cell panel: Shows the inhibition of cell proliferation of each cancer cell line by PTX-βGlcNAc. Cytotoxicity test results using a JFCR39 cell panel: Shows the inhibition of cell proliferation of each cancer cell line by DTX-βGlcNAc. Cytotoxicity test results using a JFCR39 cell panel: shows the inhibition of cell proliferation of each cancer cell line by CBZ-βGlcNAc. Shows the results of verifying the therapeutic effect using a peritoneal dissemination model with DOX-βGlcNAc. Shows the results of verifying the therapeutic effect using a peritoneal dissemination model with Mel-βGlcNAc. Shows the results of verifying the therapeutic effect using a peritoneal dissemination model with MMAE-βGlcNAc. Shows the results of verifying the therapeutic effect using a peritoneal dissemination model with PTX-βGlcNAc.1 shows the results of verifying the therapeutic effect of ETP-βGlcNAc using a peritoneal dissemination model. 2 shows the results of verifying the therapeutic effect of SN38-βGlcNAc using a peritoneal dissemination model.

[0013] In this specification, unless otherwise specified, an "alkyl group" or the alkyl portion of a substituent containing an alkyl portion (for example, an alkoxy group) means, for example, an alkyl group having 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, and more preferably about 1 to 3 carbon atoms, which is linear, branched, cyclic, or a combination thereof. More specific examples of the alkyl group include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, a cyclopropyl group, an n-butyl group, a sec-butyl group, an isobutyl group, a tert-butyl group, a cyclopropylmethyl group, an n-pentyl group, an n-hexyl group, and the like.

[0014] As used herein, the term "halogen atom" may refer to any of a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom, and is preferably a fluorine atom, a chlorine atom, or a bromine atom.

[0015] 1. Compounds Represented by General Formula (I) or Salts Thereof One embodiment of the present invention is a compound represented by the following general formula (I) or a pharmaceutically acceptable salt thereof (hereinafter also referred to as "compounds of the present invention"). Y-T-X １ (I) (Wherein, Y is -OL １ (1) or -NH-L ２ (2) is selected from L １ is a partial structure of a saccharide, where the partial structure of a saccharide is a structure obtained by removing one hydroxyl group from a saccharide, and L ２ is a monovalent substituent containing an amino acid residue or an oligopeptide residue; T is a linker or bond; X １ is at least one selected from the group consisting of: (1) a binding residue of a biologically active compound having one or more functional groups selected from the group consisting of an aliphatic hydroxyl group, an aromatic hydroxyl group, an amino group, and a carboxyl group; (2) an antibody; and (3) a ligand selected from a small molecule, a peptide, an aptamer, or a hormone-drug conjugate, with the proviso that X １at least one of the following is a binding residue of the biologically active compound; the biologically active compound is an anti-cancer drug; and n is an integer of 1 or greater.

[0016] That is, the present inventors conceived the idea that by introducing a substrate moiety that targets the cancer cell-specific enzyme activity discovered by screening of a fluorescent probe into the molecules of various anticancer drugs, including SN38, it might be possible to develop new prodrug-type anticancer drugs based on anticancer drugs such as SN38, which would selectively exhibit efficacy at cancer sites and have reduced side effects (see Figure 1 ), and conducted extensive studies. As a result, they found that the compound represented by the above general formula (I) can be released and selectively exhibit efficacy at cancer sites by cleaving the enzyme recognition site and / or linker site due to the cancer cell-specific enzyme activity, and is therefore useful as a novel prodrug-type anticancer drug.

[0017] Here, in general formula (I), Y is an enzyme recognition site, a portion of which is cleaved by cancer cell-specific enzymatic activity to release an anticancer drug molecule. As described above, SN38 is primarily used as irinotecan (CPT-11), a compound endowed with a hydrolysis site for carboxylesterase (CES). CPT-11 is a prodrug designed to release SN38 upon degradation of its side chain by CES. However, CES is not an enzyme specific to cancer sites and is highly expressed in various organs (e.g., the large intestine and liver), making it unable to release SN38 specifically at cancer sites, resulting in organ damage. Such problems are typically solved by, for example, repurposing known prodrug substrates used in other anticancer drugs. In contrast, the present invention utilizes a substrate site that targets cancer cell-specific enzymatic activity, discovered by screening of a fluorescent probe, as a substrate for a prodrug anticancer drug. According to the inventors' understanding, this is the first time that a prodrug anticancer drug has been developed using such a method.

[0018] Y can be selected depending on the type of target enzyme. When the cancer biomarker enzyme that is the target enzyme of the prodrug-type anticancer drug is a glycosidase, Y is selected from groups derived from saccharides, and when the target enzyme is a peptidase, Y is selected from groups derived from amino acids or groups containing amino acids.

[0019] In the general formula (I), Y is -OL １ (1) or -NH-L ２ (2) is selected from:

[0020] In formula (1), L １ is a partial structure of a sugar. １ The partial structure of the sugar is L １ Together with the O to which it is bound, it forms a sugar or a part of a sugar. Also, a partial structure of a sugar is a structure in which one hydroxyl group is removed from a sugar.

[0021] Examples of saccharides include β-D-glucose, β-D-galactose, β-L-galactose, β-D-xylose, α-D-mannose, β-D-fucose, α-L-fucose, β-L-fucose, β-D-arabinose, β-L-arabinose, β-D-N-acetylglucosamine, β-D-N-acetylgalactosamine, etc., with β-D-galactose, β-D-N-acetylgalactosamine, α-D-mannose, and α-L-fucose being preferred. That is, in investigating the present invention, the present inventors screened human lung cancer specimens for fluorescent probes for detecting glycosidase activity. As a result, they found that excellent prodrug anticancer drugs can be provided by introducing into an anticancer drug molecule a specific sugar selected from β-D-galactose (β-D-Gal), β-D-N-acetylgalactosamine (β-D-GlcNAc), α-D-mannose (α-D-Man), and α-L-fucose (α-L-Fuc) as a substrate moiety that targets the cancer cell-specific enzyme activity that has been confirmed to be effective as a lung cancer detection probe.

[0022] Formula (2): -NH-L ２ In this case, L ２is a monovalent substituent containing an amino acid residue or an oligopeptide residue.

[0023] As used herein, the term "amino acid" refers to any compound having both an amino group and a carboxyl group, including natural and unnatural amino acids. Neutral, basic, or acidic amino acids may be used. In addition to amino acids that function as neurotransmitters and other transmitters, amino acids that are components of polypeptide compounds such as physiologically active peptides (including dipeptides, tripeptides, tetrapeptides, and oligopeptides) and proteins may also be used, such as α-amino acids, β-amino acids, and γ-amino acids. Optically active amino acids are preferably used as amino acids. For example, while either D- or L-amino acids may be used for α-amino acids, it may be preferable to select optically active amino acids that function in living organisms.

[0024] As used herein, the term "amino acid residue" refers to a structure corresponding to the partial structure remaining after removing the hydroxyl group from the carboxyl group of an amino acid. Amino acid residues include α-amino acid residues, β-amino acid residues, and γ-amino acid residues. Preferred amino acid residues include the γ-glutamyl group of a GGT substrate, a dipeptide of a DPP4 substrate (a dipeptide consisting of an amino acid and proline), and dipeptides such as KK, QA, and TF.

[0025] As used herein, "peptide" refers to a structure in which two or more amino acids are linked by peptide bonds. "Peptide residue" refers to a structure corresponding to the partial structure remaining after removing the hydroxyl group from the carboxyl group of the C-terminal amino acid constituting the peptide. Preferred peptides include the above-mentioned DPP4 substrate dipeptide (a dipeptide consisting of amino acid-proline; here, the amino acid is, for example, glycine, glutamic acid, or proline), KK, which is the target sequence of PSA, and the like.

[0026] The N-terminus of the monovalent substituent containing an amino acid residue or an oligopeptide residue may be protected. Examples of the protecting group include an acetyl group, a glutaryl group, a succinyl group, a tert-butoxycarbonyl group, and a benzyloxycarbonyl group, but substituents other than these may also be used.

[0027] In the present invention, Y has a structure selected from the following:

[0028] Also, in one preferred aspect of the present invention, Y has a structure selected from the following:

[0029] In general formula (1), T represents a linker or a bond. When T is a bond, Y is connected to X without a linker. １ This refers to the case where the compound is combined with the compound.

[0030] The linker is X １ and Y, or a site where two or more divalent groups are linked, or a combination thereof. A variety of linkers can be used, but in the present invention, a linker that is cleaved after an enzymatic reaction (a so-called self-cleaving linker) is preferred, and examples of such linkers include a benzyl group-type linker, a carbamate group-type linker, a carbonate group-type linker, and a combination of two or more of these.

[0031] Benzyl group-type linkers include those where Y is -OL １ When the oxygen atom is —O—Ph—CH ２ -O- ＊ and Y is -NH-L ２ When the group is —NH—Ph—CH ２ -O- ＊ Here, Ph represents a substituted or unsubstituted benzene ring, * represents X １ For carbamate group type linkers, Y represents the bonding direction to -OL １ When the formula is —O—CO—NH—, it is combined with the oxygen atom to form —O—CO—NH— ＊As a carbonate group type linker, Y may be -OL １ When the oxygen atom is —O—CO—O—, the ＊ can be configured.

[0032] In addition, a linker in which a benzyl group type and a carbamate group type are combined, or a linker in which a benzyl group type and a carbonate group type are combined can also be used. １ When the oxygen atom is —O—Ph—CH ２ —O—CO—NH— ＊ and Y is -NH-L ２ When the group is —NH—Ph—CH ２ —O—CO—NH— ＊ For example, Y can be -OL １ When the oxygen atom is —O—Ph—CH ２ -O-CO-O- ＊ and Y is -NH-L ２ When the group is —NH—Ph—CH ２ -O-CO-O- ＊ Here, Ph represents a substituted or unsubstituted benzene ring, * represents X １ represents the direction of bonding.

[0033] In addition, the linker that can be used in the present invention includes two or more X １ Also included are those that can be combined with

[0034] In one preferred aspect of the present invention, the linker represented as T in general formula (1) is selected from the group consisting of the following structures (a) to (f) and combinations of two or more thereof:

[0035] In the above formulas (a), (d) to (f), R １ ~R ４may be the same or different, and each independently represents hydrogen, a methyl group, a methoxy group, —COOMe (Me represents a methyl group), —NHMe, F, Cl, Br, and NO ２ is selected from the group consisting of:

[0036] R in formula (e) ５ is hydrogen or an alkyl group having 1 to 3 carbon atoms. ６ is independently in each occurrence hydrogen or an alkyl group having 1 to 3 carbon atoms.

[0037] For example, non-limiting examples of linkers having the structure (a) include the following:

[0038] In addition, R in the formulas (a), (d) to (f) １ ~R ４ At least one of may be a linking group selected from the following: Here, in the linkers represented by the formulae (a), (d) to (f), R １ ~R ４ When at least one of is a linking group, the linker can be connected to X １ can be combined with

[0039] In the formulae (a) to (f), * indicates the direction of bonding to X. However, when T has a structure that is a combination of two or more of the formulae (a) to (f), X １ The * symbol for one or more linkers other than the linker (also referred to as "linker A") that binds to X can represent the direction of binding to the linker A. １ When at least one of the above is an antibody or a ligand, the linkers of formulas (a) to (f) and the linking groups a1 to a3 can be bonded in the direction indicated by * to a linker having a binding site that can be used as a point of attachment to an antibody or ligand, as described below.

[0040] Non-limiting examples of cases where T has a structure that is a combination of two or more of the formulae (a) to (f) include, for example, cases where T has a structure that is a combination of two formulae selected from the formulae (a) to (f), such as (1) a case where linker A is represented by formula (a) and another linker is represented by any of (a) to (f), and a case where linker A is a linker represented by formula (d) and another linker is represented by any of (a) to (f), but are not limited to these.

[0041] When T has a structure that is a combination of two formulas selected from formulas (a) to (f), non-limiting specific structures include the following:

[0042] In the linkers having the structures (b1) to (b3) above, all of the benzene rings are unsubstituted, but at least one of the benzene rings is R １ ~R ４ In addition, a structure in which T is a combination of three or more formulas selected from the formulas (a) to (f) can be constructed in the same manner as the above structures (b1) to (b3).

[0043] In addition, the linker represented by T may include, as described above, R １ ~R ４ In addition, in the case of a linker having a structure that is a combination of two or more of the formulae (a) to (f), the linker moieties of the formulae (a), (d) to (f) are also included. １ ~R ４ At least one of them may have a linking group selected from (a1) to (a3).

[0044] These linkers can also be linked to X via the linking group. １ That is, the compound of the present invention having such a linker structure can be bonded to two or more X １ R in the formulas (a), (d) to (f) can be bonded to １ ~R ４ Of these, X１ The number of linking groups that can be bonded to may be 1 or more, 2 or more, or 3 or more. The position at which the linking group is introduced may be an R that is ortho-position relative to the direction of bonding to the enzyme recognition site (the bond of the benzene ring) in the linkers of the formulae (a), (d) to (f). １ , R ２ In one aspect of the present invention, in the formulae (a), (d) to (f), R １ , R ２ A linking group selected from (a1) to (a3) ​​is introduced into either one or both of the above.

[0045] Non-limiting examples of the structure of the linker having a linking group selected from the above (a1) to (a3) ​​include the following. Here, the linker having the structure (c1) may have another linker bonded to the bonding side of the benzene ring.

[0046] In one preferred aspect of the invention, Y is —NH—L ２ In this case, the linker represented by T in general formula (1) is preferably formula (a), (d), (e), or (f), or a combination of one of these with any one or more selected from formulas (a) to (f).

[0047] In the compound of the present invention, R in the formulas (a), (d) to (f) １ ~R ４ At least one of X may be a linker with a binding site that can be used as a point of attachment to an antibody or ligand. １ (2) an antibody; (3) a ligand selected from a small molecule, a peptide, an aptamer, or a hormone-drug conjugate; or both, but X １ When such a molecule is included as R １ ~R ４ Preferably, at least one of X is a linker having an appropriate binding site capable of binding to an antibody or a ligand. １When the compound includes (2) an antibody, (3) a ligand selected from a small molecule, peptide, aptamer, or hormone-drug conjugate, or both, the linker of formulas (a) to (f) may be bonded to the antibody or ligand, but the linkers of formulas (a) to (f) and the linking groups of (a1) to (a3) ​​may further have a linker having a suitable binding site capable of binding to the antibody or ligand. (In other words, the linkers of formulas (a) to (f) and the linking groups of (a1) to (a3) ​​may be bonded to a linker having a suitable binding site capable of binding to the antibody or ligand in the direction indicated by *.) Examples of such linkers include polyethylene glycol, alkylene, peptide, and combinations thereof. Also, known linkers used in conjugates of cell-binding molecules (cell-binding ligands or receptor analogs) and cytotoxic agents, antibody-drug conjugates, etc. may be used.

[0048] In general formula (I), X １ represents at least one selected from the group consisting of: (1) a binding residue of a biologically active compound having one or more functional groups selected from the group consisting of an aliphatic hydroxyl group, an aromatic hydroxyl group, an amino group, and a carboxyl group; (2) an antibody; and (3) a ligand selected from a small molecule, a peptide, an aptamer, or a hormone-drug conjugate. In this specification, the binding residue of a biologically active compound refers to a biologically active compound having a functional group selected from the group consisting of -OL, where Y is a hydroxyl group, an aromatic hydroxyl group, an amino group, and a carboxyl group. １ or -NH-L ２ When Y is directly bonded to X (when bonded without a linker) or when bonded to a linker, it means the part of the molecular structure of the biologically active compound excluding the functional group involved in the bond. １ When Y is bonded to the group represented by formula (1): -OL １ When the oxygen atom and Y are represented by the formula (2): -NH-L ２ In other words, the bound residue of the biologically active compound is a residue obtained by removing an aliphatic hydroxyl group or an aromatic hydroxyl group from the biologically active compound (where Y is -OL). １and a residue obtained by removing a hydroxyl group from a carboxy group of a biologically active compound, or a residue obtained by removing an amino group (when Y is -NH-L ２ In addition, when Y is X １ When binding to a biologically active compound, depending on the binding group (preferably, an oxygen atom or NH) at the end of the linker, the binding residue of the biologically active compound is a residue obtained by removing an aliphatic hydroxyl group or an aromatic hydroxyl group from the biologically active compound, a residue obtained by removing a hydroxyl group from a carboxyl group of the biologically active compound, or a residue obtained by removing an amino group.

[0049] X １ The biologically active compound in the binding residue of the biologically active compound represented by the formula (I) is preferably an antitumor active substance, and more preferably an anticancer agent.

[0050] The biologically active compounds that can be used in the present invention are preferably selected from the following anti-cancer agents:

[0051] Among the above anticancer agents, in the present invention, it is more preferable to use SN38, nogitecan, exatecan, etoposide, doxorubicin, epirubicin, melphalan, MMAE, MMAF, paclitaxel, docetaxel, or cabazitaxel as the biologically active compound, and it is particularly preferable to use SN38, etoposide, doxorubicin, melphalan, MMAE, or paclitaxel.

[0052] When the biologically active compound has two or more functional groups selected from the group consisting of an aliphatic hydroxyl group, an aromatic hydroxyl group, an amino group, and a carboxyl group, it is preferable to introduce Y as one of the substituents capable of sufficiently inhibiting pharmaceutical activity when a substrate moiety is introduced. For example, when SN38 is used as the biologically active compound, it is preferable to introduce and bind a partial structure of a saccharide of L to the position of the hydroxyl group bonded to the terminal benzene ring. Furthermore, when paclitaxel is used as the biologically active compound, it is preferable to introduce and bind a substrate moiety to the hydroxyl group of the terminal side chain structure or the hydroxyl group at position 9.

[0053] In the compound represented by formula (I), X １ At least one of the groups is a binding moiety for a biologically active compound.

[0054] The compounds of the present invention include X １ Examples of such compounds include compounds containing a binding residue of one biologically active compound that is an anticancer drug, and compounds containing binding residues of two or more (preferably two or three) biologically active compounds that are anticancer drugs. When containing binding residues of two or more (preferably two or three) biologically active compounds that are anticancer drugs, the anticancer drugs may be the same or different (in whole or in part).

[0055] The compound of the present invention is also １ The compound of the present invention may include (2) an antibody, (3) a ligand selected from a small molecule, a peptide, an aptamer, or a hormone-drug conjugate, or both. Here, the ligand selected from an antibody, a small molecule, a peptide, an aptamer, or a hormone-drug conjugate that can be used in the compound of the present invention is a tumor-associated antibody or ligand, or a diseased tissue-associated antibody or ligand.

[0056] Such ligands include, for example, the following cell-binding ligands or receptor analogs: LB01 (PMSA ligand conjugate), LB02 (folate receptor conjugate), LB03 (somatostatin receptor conjugate), LB04 (octreotide, somatostatin analog receptor conjugate), LB05 (lanreotide, somatostatin analog receptor conjugate), LB06 (CAIX receptor conjugate), LB07 (CAIX receptor conjugate), LB08 (luteinizing hormone-releasing hormone (LH-RH) ligand), LB09 (luteinizing hormone-releasing hormone (LH-RH) ligand), LB10 (luteinizing hormone-releasing hormone (LH-RH) ligand), LB11 (luteinizing hormone-releasing hormone (LH-RH) receptor conjugate), LB12 (luteinizing hormone-releasing hormone (LH-RH) receptor conjugate), LB13 (luteinizing hormone-releasing hormone (LH-RH) ligand), LB14 (luteinizing hormone-releasing hormone (LH-RH) receptor conjugate), LB15 (luteinizing hormone-releasing hormone (LH-RH) receptor conjugate), LB16 (CAIX receptor conjugate), LB17 (CAIX receptor conjugate), LB18 (luteinizing hormone-releasing hormone (LH-RH) ligand), LB19 (luteinizing hormone-releasing hormone (LH-RH) receptor conjugate), LB20 (luteinizing hormone-releasing hormone (LH-RH) receptor conjugate), LB21 (luteinizing hormone-releasing hormone (LH-RH) receptor conjugate), LB22 (luteinizing hormone-releasing hormone (LH-RH) receptor conjugate), LB23 (luteinizing hormone-releasing hormone (LH-RH) receptor conjugate), LB24 (luteinizing hormone-releasing hormone (LH-RH) receptor conjugate), LB25 (luteinizing hormone-releasing hormone (LH-RH) receptor and GnRH conjugate), LB09 (luteinizing hormone-releasing hormone (LH-RH) and GnRH ligand conjugate), LB10 (GnRH antagonist, abarelix conjugate), LB11 (cobalamin, VB12 analog conjugate), LB12 (gastrin-releasing peptide receptor (GRPr), MBA conjugate), LB13 (αvβ3 integrin receptor, cyclic RGD pentapeptide conjugate), LB14 (heterobivalent peptide ligand of VEGF receptor conjugate), LB15 (neuromedin B conjugate), LB16 (G protein-coupled receptor, bombesin conjugate), and LB17 (Toll-like receptor, TLR2 conjugate).

[0057] Examples of tumor-associated antibodies include anti-HER2 antibodies, anti-TROP2 antibodies, anti-claudin antibodies, and anti-PD-L1 antibodies.

[0058] Specific anti-human PD-L1 monoclonal antibodies useful as PD-L1 antibodies include, but are not limited to, avelumab (MSB0010718C), nivolumab (BMS-936558), MPDL3280A (an IgG1 engineered anti-PD-L1 antibody), BMS-936559 (a fully human anti-PD-L1, IgG4 monoclonal antibody), MEDI4736 (an engineered IgG1κ monoclonal antibody with a triple mutation in the Fc domain to ablate antibody-dependent cell-mediated cytotoxicity), and antibodies comprising the heavy and light chain variable regions of SEQ ID NO:24 and SEQ ID NO:21, respectively, of WO 2013 / 019906.

[0059] The term "anti-HER2 antibody" refers to an antibody that specifically binds to HER2 (Human Epidermal Growth Factor Receptor Type 2; ErbB-2), and preferably has the activity of being internalized into HER2-expressing cells upon binding to HER2. Examples of anti-HER2 antibodies include trastuzumab (U.S. Pat. No. 5,821,337) and pertuzumab (WO 01 / 00245), with trastuzumab being preferred.

[0060] The term "anti-TROP2 antibody" refers to an antibody that specifically binds to TROP2 (TACSTD2: Tumor-associated calcium signal transducer 2; EGP-1), and preferably has the activity of being internalized into TROP2-expressing cells upon binding to TROP2. An example of an anti-TROP2 antibody is hTINA1-H1L1 (WO 2015 / 098099).

[0061] Claudins are integral membrane proteins located at tight junctions between epithelia and endothelia. Claudins are predicted to have four transmembrane domains with two extracellular loops, and N- and C-termini located in the cytoplasm. The claudin (CLDN) family of transmembrane proteins plays an important role in maintaining tight junctions in epithelia and endothelia and may also be involved in cytoskeletal maintenance and cell signaling.

[0062] The claudin 18 (CLDN18) molecule has four hydrophobic transmembrane regions and two extracellular loops (loop 1 surrounded by hydrophobic regions 1 and 2, and loop 2 surrounded by hydrophobic regions 3 and 4). Known anti-claudin antibodies can be used in the compounds of the present invention.

[0063] The compound of the present invention is １When the compound of the present invention contains the tumor-associated antibody, it can also be used as a targeted drug delivery agent, so-called antibody-drug conjugate (ADC). When the compound of the present invention contains an antibody against a tumor-associated antigen, the antibody can serve as a highly potent targeting vehicle for the cytotoxic agent (the anticancer drug in the compound of the present invention), i.e., the "payload." Generally, after administration, the ADC distributes throughout the patient's body and binds to the antigen on the surface of tumor cells. The antibody-antigen complex is then internalized by the cell and directed to lysosomes via endogenous intracellular transport pathways. After reaching the lysosome, the ADC is degraded, thereby releasing its toxic cargo. The free toxin can then bind to its intracellular target, thus inducing apoptosis and cancer cell death. Because the antibody should bind and deliver the toxin only to cancer cells that express the antigen, healthy cells are largely spared. By using a prodrug-type anticancer drug containing the compound of the present invention as an ADC, it can be selectively delivered to cancer cells. Once taken up into the cancer cells, the enzyme recognition site and / or linker site are cleaved by an enzyme activity specific to the cancer cells, allowing the anticancer drug to be released with higher selectivity within the cancer cells.

[0064] The compound of the present invention is １ When R in the above formulas (d) to (f) includes an antibody; a ligand selected from a small molecule, a peptide, an aptamer, or a hormone-drug conjugate; or both, the linker represented by T in general formula (I) preferably has a binding site that can be used as a point of attachment to the antibody or the ligand. １ ~R ４ At least one of X may be a linker having a binding site that can be used as a point of attachment to an antibody or ligand. １The compound may contain (2) an antibody, (3) a ligand selected from a small molecule, peptide, aptamer, or hormone-drug conjugate, or both, and may be linked to a linker having a suitable binding site that can bind to the antibody or ligand in the direction indicated by the linker * in formulas (a) to (f). Examples of such linkers include polyethylene glycol, alkylene, peptide, and combinations thereof. Also, known linkers used in conjugates of cell-binding molecules (cell-binding ligands or receptor analogs) and cytotoxic agents, antibody-drug conjugates, and the like, may be used. The ligand can be linked to a cysteine, lysine, glutamine, or other reactive complex site on the ligand via a binding site.

[0065] In general formula (I), n is an integer of 1 or more, and may be an integer of 2 or more (preferably 2 or 3).

[0066] In one preferred aspect of the invention, n is 1.

[0067] Non-limiting examples of the compounds of the present invention are shown below, but the compounds of the present invention are not limited to these.

[0068] Unless otherwise specified, the compounds represented by general formula (I) also include stereoisomers such as tautomers, geometric isomers (e.g., E-isomer, Z-isomer, etc.), and enantiomers. That is, when the compound represented by general formula (I) contains one or more asymmetric carbon atoms, the stereochemistry of the asymmetric carbon atoms can each independently take either the (R) or (S) form, and the derivative may exist as a stereoisomer such as an enantiomer or diastereoisomer. Therefore, any pure stereoisomer, any mixture of stereoisomers, or a racemate can be used as the active ingredient of the prodrug-type anticancer agent of the present invention described below, and all of these are included in the scope of the compound of the present invention.

[0069] The method for producing the compound represented by general formula (I) is not particularly limited, but the synthesis methods for representative compounds among the compounds included in general formula (I) are specifically shown in the Examples of this specification. Those skilled in the art can produce the compounds included in formula (I) by appropriately changing or modifying the starting materials, reaction reagents, reaction conditions, etc. as necessary, while referring to the Examples of this specification.

[0070] 2. Prodrug-Type Anticancer Agents Another embodiment of the present invention is a prodrug-type anticancer agent comprising a compound of general formula (I) or a pharmaceutically acceptable salt thereof (hereinafter also referred to as "prodrug-type anticancer agent of the present invention"). Another embodiment of the present invention is a prodrug-type anticancer agent comprising a compound of general formula (I) or a pharmaceutically acceptable salt thereof, which acts cell-selectively by virtue of a cancer cell-specific enzyme activity.

[0071] Cancer cell-specific enzymes include glycosidases and peptidases. Examples of glycosidases include β-galactosidase, β-glucosidase, α-mannosidase, α-fucosidase, β-N-acetylgalactosaminidase, and β-hexosaminidase (a dimer of β-hexosaminidase subunit A (HEXA; hereinafter abbreviated as "A") and β-hexosaminidase subunit B (HEXB; hereinafter abbreviated as "B"), and composed of AA, AB, BB, etc.). Examples of peptidases include γ-glutamyltranspeptidase (GGT), dipeptidyl peptidase IV (DPP-IV), calpain, puromycin-sensitive aminopeptidase (PSA), aminopeptidase N (APN), and cathepsin.

[0072] Another embodiment of the present invention is a pharmaceutical composition for treating or preventing cancers such as breast cancer, esophageal cancer, lung cancer, stomach cancer, colon cancer, small intestine cancer, pancreatic cancer, liver cancer, head and neck cancer, oral cancer, ovarian cancer, brain tumor, kidney cancer, prostate cancer, and skin cancer, which comprises a compound of general formula (I) or a pharmaceutically acceptable salt thereof (hereinafter also referred to as the "pharmaceutical composition of the present invention").

[0073] Another embodiment of the present invention is a method for treating cancer such as breast cancer, esophageal cancer, lung cancer, stomach cancer, colon cancer, small intestine cancer, pancreatic cancer, liver cancer, head and neck cancer, oral cancer, ovarian cancer, brain tumor, kidney cancer, prostate cancer, skin cancer, etc. in a mammal, particularly a human, which comprises administering to a mammal in need of such treatment an effective amount of the compound of the present invention represented by general formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a compound represented by general formula (I) or a pharmaceutically acceptable salt thereof.

[0074] The prodrug-type anticancer agent or pharmaceutical composition of the present invention may contain not only the compound represented by general formula (I) but also a salt thereof, or a solvate or hydrate thereof. The salt is not particularly limited as long as it is a pharmaceutically acceptable salt, and examples thereof include base addition salts, acid addition salts, and amino acid salts. Examples of base addition salts include alkaline earth metal salts such as sodium salt, potassium salt, calcium salt, and magnesium salt, ammonium salt, and organic amine salts such as triethylamine salt, piperidine salt, and morpholine salt. Examples of acid addition salts include mineral acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate; and organic acid salts such as methanesulfonic acid, benzenesulfonic acid, paratoluenesulfonic acid, acetic acid, propionate, tartaric acid, fumaric acid, maleic acid, malic acid, oxalic acid, succinic acid, citric acid, benzoic acid, mandelic acid, cinnamic acid, lactic acid, glycolic acid, glucuronic acid, ascorbic acid, nicotinic acid, and salicylic acid. Examples of amino acid salts include glycine salts, aspartates, glutamates, etc. Metal salts such as aluminum salts may also be used.

[0075] The type of solvent that forms the solvate is not particularly limited, but examples include solvents such as ethanol, acetone, and isopropanol.

[0076] The prodrug-type anticancer agent of the present invention can be expected to be highly effective against a wide range of cancers, including breast cancer, esophageal cancer, lung cancer, stomach cancer, colon cancer, small intestine cancer, pancreatic cancer, liver cancer, head and neck cancer, oral cancer, ovarian cancer, brain tumors, kidney cancer, prostate cancer, and skin cancer, among others.

[0077] Although the prodrug-type anticancer agent or pharmaceutical composition of the present invention may be administered as the active ingredient, that is, the compound represented by general formula (I) or a pharmaceutically acceptable salt, hydrate, or solvate thereof, itself, it is generally desirable to administer it in the form of a pharmaceutical composition containing the above substance as the active ingredient and one or more formulation additives. The term "composition," as in pharmaceutical composition, encompasses not only a product containing an active ingredient and an inactive ingredient (a pharmaceutically acceptable excipient) that constitutes a carrier, but also any product that occurs directly or indirectly as a result of the association, complexation, or aggregation of any two or more components, or as a result of the dissociation of one or more components, or as a result of another type of reaction or interaction of one or more components.

[0078] As the active ingredient of the prodrug-type anticancer agent or pharmaceutical composition of the present invention, two or more of the above compounds can be used in combination.

[0079] Furthermore, the prodrug-type anticancer agent or pharmaceutical composition of the present invention can also be used as a combination drug by combining the active ingredient, the compound represented by general formula (I) or a pharmaceutically acceptable salt, hydrate, or solvate thereof, with an existing anticancer agent. Existing anticancer agents that can be used include those known in the technical field, such as methotrexate, doxorubicin, cisplatin, and anti-PD-1 antibodies. Anti-PD-1 antibodies include, but are not limited to, avelumab (MSB0010718C), nivolumab (BMS-936558), MPDL3280A (an IgG1-engineered anti-PD-L1 antibody), BMS-936559 (a fully human anti-PD-L1, IgG4 monoclonal antibody), MEDI4736 (an engineered IgG1κ monoclonal antibody with a triple mutation in the Fc domain to ablate antibody-dependent cell-mediated cytotoxicity), and antibodies comprising the heavy and light chain variable regions of SEQ ID NO:24 and SEQ ID NO:21, respectively, of WO 2013 / 019906.

[0080] The type of prodrug-type anticancer agent or pharmaceutical composition of the present invention is not particularly limited, and examples of dosage forms include tablets, capsules, granules, powders, syrups, suspensions, suppositories, ointments, creams, gels, patches, inhalants, and injections. These formulations are prepared according to conventional methods. Liquid formulations may be dissolved or suspended in water or other suitable solvents before use. Tablets and granules may also be coated by known methods. Injections are prepared by dissolving the compound of the present invention in water, but may also be dissolved in physiological saline or glucose solution as needed, and buffers and preservatives may also be added. They may be provided in any dosage form for oral or parenteral administration. For example, it can be prepared as a pharmaceutical composition for oral administration in the form of granules, fine granules, powders, hard capsules, soft capsules, syrups, emulsions, suspensions, or solutions, or as a pharmaceutical composition for parenteral administration in the form of injections for intravenous, intramuscular, or subcutaneous administration, drip infusions, transdermal absorption preparations, transmucosal absorption preparations, nasal drops, inhalants, suppositories, etc. Injections and drip infusions can also be prepared in a powdered dosage form, such as a lyophilized form, and dissolved in an appropriate aqueous medium, such as physiological saline, before use. It is also possible to administer sustained-release preparations coated with a polymer or the like directly into the brain.

[0081] The type of formulation additive used in the production of the prodrug-type anticancer agent or pharmaceutical composition of the present invention, the ratio of the formulation additive to the active ingredient, or the method for producing the pharmaceutical composition can be appropriately selected by those skilled in the art depending on the form of the composition. As the formulation additive, an inorganic or organic substance, or a solid or liquid substance can be used, and generally, it can be blended in an amount of 1 to 90% by weight based on the weight of the active ingredient. Specific examples of such substances include lactose, glucose, mannitol, dextrin, cyclodextrin, starch, sucrose, magnesium aluminometasilicate, synthetic aluminum silicate, sodium carboxymethylcellulose, hydroxypropyl starch, calcium carboxymethylcellulose, ion exchange resins, methylcellulose, gelatin, gum arabic, hydroxypropyl cellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, light anhydrous silicic acid, magnesium stearate, talc, tragacanth, bentonite, Veegum, titanium oxide, sorbitan fatty acid esters, sodium lauryl sulfate, glycerin, fatty acid glycerin esters, purified lanolin, glycerogelatin, polysorbate, macrogol, vegetable oils, wax, liquid paraffin, white petrolatum, fluorocarbons, nonionic surfactants, propylene glycol, and water.

[0082] To prepare solid formulations for oral administration, the active ingredient is mixed with excipients such as lactose, starch, crystalline cellulose, calcium lactate, and anhydrous silicic acid to form a powder, or, if necessary, with binders such as sucrose, hydroxypropyl cellulose, and polyvinylpyrrolidone, and disintegrants such as carboxymethylcellulose and calcium carboxymethylcellulose, followed by wet or dry granulation to form granules. To prepare tablets, these powders and granules can be compressed directly or with the addition of lubricants such as magnesium stearate and talc. These granules or tablets can be coated with enteric-coated bases such as hydroxypropylmethylcellulose phthalate and methacrylic acid-methyl methacrylate polymer to form enteric-coated formulations, or coated with ethylcellulose, carnauba wax, and hydrogenated oil to form sustained-release formulations. To prepare capsules, the powder or granules can be filled into hard capsules, or the active ingredient can be dissolved directly or in glycerin, polyethylene glycol, sesame oil, olive oil, etc., and then coated with a gelatin membrane to form soft capsules.

[0083] To prepare an injection, the active ingredient may be dissolved in distilled water for injection, if necessary, together with a pH adjuster such as hydrochloric acid, sodium hydroxide, lactose, lactic acid, sodium, sodium monohydrogen phosphate, sodium dihydrogen phosphate, etc., and an isotonic agent such as sodium chloride or glucose, and then sterile filtered and filled into an ampule, or mannitol, dextrin, cyclodextrin, gelatin, etc. may be added, followed by vacuum freeze-drying to produce an injection that is dissolved before use. Alternatively, the active ingredient may be emulsified in water with lecithin, polysorbate 80, polyoxyethylene hydrogenated castor oil, etc. to produce an emulsion for injection.

[0084] The dosage and frequency of administration of the prodrug-type anticancer agent or pharmaceutical composition of the present invention are not particularly limited, and can be appropriately selected at the discretion of a physician depending on conditions such as the purpose of preventing and / or treating the worsening / progression of the disease to be treated, the type of disease, the patient's weight and age, and the severity of the disease. Generally, the daily oral dose for adults is about 0.01 to 1000 mg (weight of active ingredient), and can be administered once a day or in divided doses several times a day, or every few days. When used as an injection, it is desirable to administer a daily dose of 0.001 to 100 mg (weight of active ingredient) to an adult continuously or intermittently.

[0085] 3. Novel Fluorescent Probe for Detecting Lung Cancer In conducting research and development of the prodrug anticancer drug of the present invention, the present inventors screened fluorescent probes for detecting glycosidase activity in human lung cancer specimens and found that a previously unknown fluorescent probe is highly effective as a probe for detecting lung cancer. That is, another embodiment of the present invention is a fluorescent probe for detecting lung cancer, comprising a compound represented by the following general formula (II) or a salt thereof (hereinafter also referred to as the "fluorescent probe of the present invention").

[0086] In general formula (I), R １ When present, represents the same or different monovalent substituents present on the benzene ring. Examples of the monovalent substituent include halogen and optionally substituted alkyl groups.

[0087] m is an integer from 0 to 4. In one preferred aspect of the invention, m is 0 and R １ does not exist. In other words, the benzene ring bonded to the xanthene skeleton is an unsubstituted benzene ring.

[0088] In general formula (I), R ２ and R ３ R each independently represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom. ２ and R ３When R represents an alkyl group, the alkyl group may contain one or more halogen atoms, carboxy groups, sulfonyl groups, hydroxyl groups, amino groups, alkoxy groups, etc., and for example, R ２ or R ３ The alkyl group represented by R may be a halogenated alkyl group, a hydroxyalkyl group, a carboxyalkyl group, etc. ２ and R ３ are preferably each independently a hydrogen atom or a halogen atom. ２ and R ３ is a halogen atom, R ２ and R ３ In one preferred aspect of the present invention, it is preferable that both of R ２ and R ３ are both hydrogen atoms.

[0089] R ４ and R ５ each independently represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom; R ２ and R ３ This is the same as that explained for R. ４ and R ５ are preferably both hydrogen atoms.

[0090] R ６ represents a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, or a fluorinated alkyl group (fluoroalkyl group) having 1 to 5 carbon atoms. ６ The alkyl group of R is preferably a methyl group or an ethyl group. ６ The fluorinated alkyl group is, for example, —CH ２ -CF ３ , -CH ２ -CH ２ -CF ３ In one preferred aspect of the present invention, R ６ is -CH ２ -CF ３ is.

[0091] In general formula (I), R ７ and R ８When present, each independently represents an alkyl group or an aryl group having 1 to 6 carbon atoms; ７ and R ８ are each preferably independently an alkyl group having 1 to 3 carbon atoms, and R ７ and R ８ It is more preferable that both R ７ and R ８ The alkyl group represented by may contain one or more halogen atoms, carboxy groups, sulfonyl groups, hydroxyl groups, amino groups, alkoxy groups, etc., and for example, R ７ or R ８ The alkyl group represented by R may be a halogenated alkyl group, a hydroxyalkyl group, a carboxyalkyl group, etc. ７ or R ８ When represents an aryl group, the aryl group may be either a monocyclic aromatic group or a fused aromatic group, and the aryl ring may contain one or more ring-constituting heteroatoms (e.g., nitrogen atom, oxygen atom, sulfur atom, etc.). The aryl group is preferably a phenyl group. One or more substituents may be present on the aryl ring. The substituents may include, for example, one or more halogen atoms, carboxy groups, sulfonyl groups, hydroxyl groups, amino groups, alkoxy groups, etc.

[0092] In addition, when X is an oxygen atom, R ７ and R ８ does not exist.

[0093] X represents an oxygen atom, a silicon atom, or a carbon atom. In one preferred aspect of the present invention, X is an oxygen atom.

[0094] n is an integer of 1 to 3, and preferably n is 1.

[0095] L is a group of formula (1b):

[0096] The compound represented by general formula (II) can exist as an acid addition salt or a base addition salt. Examples of acid addition salts include mineral acid salts such as hydrochloride, sulfate, and nitrate, and organic acid salts such as methanesulfonate, p-toluenesulfonate, oxalate, citrate, and tartrate. Examples of base addition salts include metal salts such as sodium salt, potassium salt, calcium salt, and magnesium salt, ammonium salt, and organic amine salts such as triethylamine salt. In addition, salts with amino acids such as glycine may also be formed. The compound represented by general formula (II) or a salt thereof may exist as a hydrate or solvate, and these substances can also be used in the present invention.

[0097] The compound represented by general formula (II) may have one or more asymmetric carbon atoms depending on the type of substituent. In the present invention, stereoisomers such as optically active substances based on one or more asymmetric carbon atoms and diastereoisomers based on two or more asymmetric carbon atoms, as well as any mixture of stereoisomers and racemates can be used.

[0098] The fluorescent probe of the present invention can detect lung adenocarcinoma and lung squamous cell carcinoma, as well as small cell lung cancer, large cell lung cancer, and thymoma as a mediastinal tumor.

[0099] The fluorescent probe of the present invention can be used in any manner similar to conventionally known fluorescent probes. Typically, a compound of general formula (II) or a salt thereof is dissolved in an aqueous medium such as physiological saline or a buffer solution, or in a mixture of an aqueous medium and a water-miscible organic solvent such as ethanol, acetone, ethylene glycol, dimethyl sulfoxide, or dimethylformamide. The resulting solution is then added to an appropriate buffer solution containing cells or tissues, and the fluorescence spectrum is measured. The fluorescent probe of the present invention may also be used in the form of a composition in combination with an appropriate additive. For example, it can be combined with additives such as a buffer, solubilizer, or pH adjuster. The concentration of the compound of general formula (II) in the fluorescent probe of the present invention can be appropriately determined depending on the type of cells to be measured, the measurement conditions, etc.

[0100] Another embodiment of the present invention is a method for detecting lung cancer, comprising the steps of (a) applying the fluorescent probe of the present invention to a clinical specimen of lung cancer, and (b) measuring a fluorescent image of the clinical specimen to which the fluorescent probe has been applied. Applying the fluorescent probe to the clinical specimen of lung cancer in step (a) can be achieved, for example, by locally spraying a solution of the fluorescent probe onto the clinical specimen of lung cancer. The method for detecting lung cancer of the present invention can detect not only lung adenocarcinoma and lung squamous cell carcinoma, but also small cell lung cancer, large cell lung cancer, and thymoma as a mediastinal tumor.

[0101] Another embodiment of the present invention is a kit for detecting lung cancer, which comprises the fluorescent probe of the present invention.

[0102] In the kit, the fluorescent probe of the present invention is usually prepared as a solution, but it may also be provided as a composition in an appropriate form, such as a powder mixture, a lyophilized product, granules, tablets, or liquid, and can be applied by dissolving it in distilled water for injection or an appropriate buffer solution at the time of use.

[0103] The kit may also contain other reagents, etc., as needed. For example, additives such as solubilizing agents, pH adjusting agents, buffering agents, and isotonic agents can be used, and the amounts of these additives to be added can be appropriately selected by those skilled in the art.

[0104] 4. Companion Diagnostics In the course of developing and investigating the prodrug anticancer agent of the present invention, the present inventors screened fluorescent probes for detecting glycosidase activity in human lung cancer specimens and found that the above-described fluorescent probe of the present invention (a fluorescent probe containing a compound of formula (1b) in which L in general formula (II)) is highly effective as a novel probe for detecting lung cancer. Furthermore, as shown in Example 1, fluorescent probes incorporating β-D-Glc, β-D-Gal, or α-D-Man as L are also effective as probes for detecting lung cancer. Furthermore, depending on the substrate sequence of L, the prodrug anticancer agent of the present invention can also be used as a probe for detecting cancers other than lung cancer. Here, the prodrug anticancer agent of the present invention shares with these lung cancer detection probes the substrate site that targets the cancer cell-specific enzyme activity. Therefore, by using these probes for detecting cancers such as lung cancer in patients with lung cancer to know in advance the level of the target cancer cell-specific enzyme activity, it becomes possible to determine whether the prodrug-type anticancer drug of the present invention will be effective in the cancer patient. Therefore, these probes for detecting cancers such as lung cancer can also be used as companion diagnostic agents.

[0105] That is, another embodiment of the present invention is a companion diagnostic for diagnosing the need for cancer treatment with a prodrug-type anticancer drug comprising a compound represented by the following general formula (I) or a pharmaceutically acceptable salt thereof, the companion diagnostic comprising a compound represented by the following general formula (II) or a salt thereof (hereinafter also referred to as the "companion diagnostic of the present invention"). １ ) ｎ (I)

[0106] In formula (I), Y is selected from any of the following groups: T represents a linker or a bond. １is selected from the group consisting of: (1) a binding residue of a biologically active compound having one or more functional groups selected from the group consisting of an aliphatic hydroxyl group, an aromatic hydroxyl group, an amino group, and a carboxyl group; (2) an antibody; and (3) a ligand selected from a small molecule, a peptide, an aptamer, or a hormone-drug conjugate, provided that X １ At least one of the following is a binding residue of the biologically active compound. The biologically active compound is an anti-cancer drug. n is an integer of 1 or more.

[0107]

[0108] In formula (II), R １ When present, R represents the same or different monovalent substituents present on the benzene ring. ２ and R ３ R each independently represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom. ４ and R ５ R each independently represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom. ６ represents a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, or a fluorinated alkyl group having 1 to 5 carbon atoms. ７ and R ８ When present, each independently represents an alkyl group or an aryl group having 1 to 6 carbon atoms. When X is an oxygen atom, R ７ and R ８ does not exist. X represents an oxygen atom, a silicon atom, or a carbon atom. n is an integer of 1 to 3. L is selected from any of the following groups:

[0109] In the companion diagnostic of the present invention, T and X in general formula (I) １ The details of and n are the same as those described in detail for the compound of the present invention, and R １ ~R ８ The details of X and n are the same as those described in detail for the fluorescent probe of the present invention.

[0110] As used herein, "treatment" refers to preventing, preferably maintaining, more preferably alleviating, and even more preferably eliminating, the worsening of a certain disease or disorder (e.g., cancer) when that condition has developed, and includes the potential for symptom improvement or prophylactic effects on a patient's disease or one or more symptoms associated with the disease. Preliminary diagnosis followed by appropriate treatment is called "companion treatment," and diagnostic agents used for this purpose are called "companion diagnostic agents." Here, "companion diagnosis" typically involves measuring specific biomarkers (e.g., specific glycosidases, peptidases) using collected samples (e.g., blood or clinical samples) to determine whether treatment is appropriate (whether it can be expected to be effective).

[0111] The companion diagnostic of the present invention is used to diagnose the need for cancer treatment, preferably lung cancer treatment.

[0112] 5. Method for Treating Cancer Another embodiment of the present invention is a method for treating a patient with cancer, comprising the step of administering to the patient a therapeutically effective amount of the prodrug-type anticancer agent of the present invention or the pharmaceutical composition of the present invention (hereinafter also referred to as the "therapeutic method of the present invention").

[0113] The prodrug-type anticancer drug of the present invention can act cell-selectively due to its enzyme activity specific to cancer cells. Therefore, cancers to which the prodrug-type anticancer drug is applied are characterized by a higher expression level of the enzyme. Furthermore, in the treatment method of the present invention, preferably, the prodrug-type anticancer drug releases the anticancer drug molecule by cleavage of the enzyme recognition site and / or linker site due to the cancer cell-specific enzyme activity. That is, one aspect of the treatment method of the present invention is a method for treating cancer in which the prodrug-type anticancer drug of the present invention acts cell-selectively due to its enzyme activity specific to cancer cells, and the cancer is characterized by a higher expression level of the enzyme. Furthermore, another aspect of the treatment method of the present invention is a method for treating cancer in which the prodrug-type anticancer drug of the present invention acts cell-selectively due to its enzyme activity specific to cancer tissue, and the cancer is characterized by a higher expression level of the enzyme. Another aspect of the therapeutic method of the present invention is a method for treating cancer, in which the prodrug-type anticancer drug of the present invention releases anticancer drug molecules by cleaving the enzyme recognition site and / or linker site due to enzymatic activity specific to cancer cells. Yet another aspect of the therapeutic method of the present invention is a method for treating cancer, in which the prodrug-type anticancer drug of the present invention releases anticancer drug molecules by cleaving the enzyme recognition site and / or linker site due to enzymatic activity specific to cancer tissues.

[0114] Details of the prodrug-type anticancer agent or pharmaceutical composition of the present invention are as described in "2. Prodrug-type anticancer agent." The prodrug-type anticancer agent or pharmaceutical composition of the present invention that can be used in the cancer treatment method of the present invention can also be used as a combination drug in which the active ingredient, the compound represented by general formula (I) or a pharmaceutically acceptable salt, hydrate, or solvate thereof, is used in combination with an existing anticancer agent.

[0115] Since the prodrug-type anticancer drug used in the treatment method of the present invention acts cell-selectively due to its enzymatic activity specific to cancer cells, it is preferable that the patient (subject) to whom the prodrug-type anticancer drug is administered has enzymatic activity specific to cancer cells. It is preferable to identify whether the patient (subject) has such enzymatic activity and the level of that enzymatic activity before the start of treatment and, preferably, even during the treatment.

[0116] The level of enzyme activity is preferably identified using one or more enzyme diagnostic agents selected from visualization agents or enzyme activity reporters, such as fluorescent probes for detecting enzyme activity as described herein or other known enzyme activity diagnostic probes.

[0117] Thus, one preferred aspect of the therapeutic method of the present invention involves using one or more enzyme diagnostic agents selected from visualization agents or enzyme activity reporters to identify the level of enzymatic activity of the enzyme in a patient's biological sample and to define groups likely to respond to treatment as a function of the enzymatic activity.

[0118] Defining groups likely to respond to treatment as a function of enzyme activity can be accomplished by measuring enzyme activity in patients or in specimen samples from patients using enzyme activity-detecting fluorescent probes or other diagnostic probes.

[0119] Furthermore, the treatment method of the present invention preferably includes using an enzyme diagnostic agent to stratify a set of patients and define a subset of patients who are likely to respond to the prodrug-type anticancer drug of the present invention. Stratifying a set of patients using an enzyme diagnostic agent to define a subset of patients who are likely to respond to the prodrug-type anticancer drug can be achieved by measuring the enzyme activity of patients or specimen samples from patients using an enzyme activity-detecting fluorescent probe or other diagnostic probes.

[0120] Furthermore, to identify the level of enzyme activity, a companion diagnostic agent for diagnosing the need for cancer treatment with the above-mentioned prodrug anticancer agent of the present invention can also be used.

[0121] 6. Enzyme Recognition Site-Linker Binding Site of the Present Invention As described in detail above, the prodrug-type anticancer drug of the present invention can act cell-selectively due to its cancer cell-specific enzymatic activity. Furthermore, the prodrug-type anticancer drug of the present invention can be bound to an anticancer drug via a linker. In this case, a portion of the enzyme recognition site is cleaved by the cancer cell-specific enzymatic activity, and the linker (preferably a self-cleaving linker) is detached after the enzymatic reaction, releasing the anticancer drug molecule. As described above, the compound of the present invention can be bound to a ligand selected from an antibody, a small molecule, a peptide, an aptamer, or a hormone-drug conjugate, or both, via a linker. When a portion of the enzyme recognition site is cleaved by the cancer cell-specific enzymatic activity, and the linker (preferably a self-cleaving linker) is detached after the enzymatic reaction, these antibodies and ligands are also released from the compound of the present invention or the prodrug-type anticancer drug. Here, when an antibody or ligand has a payload (a cytotoxic drug, an enzyme inhibitor / modulator, or any molecule having biological, pharmaceutical, or diagnostic utility) other than the anticancer drug possessed by the compound of the present invention, the payload is inactive (its activity is masked) until a portion of the enzyme recognition site is cleaved by enzymatic activity, but cleavage of the linker moiety by the enzyme serves as a trigger, enabling activation of the payload. Therefore, the Y-T- portion of general formula (I) of the present invention, i.e., the portion where the enzyme recognition site and linker are bonded (hereinafter also referred to as the "enzyme recognition site-linker bond site" or "enzymatically cleavable linker"), can also be used to activate an antibody or ligand having such a payload.

[0122] Here, the enzyme recognition site in the "enzyme recognition site-linker binding site" is a substrate site that targets cancer cell-specific enzyme activity. "Cancer cell-specific" means that the enzyme activity is higher in diseased tissues, including cancer, preferably cancer cells, than in normal tissues. Methods for confirming cancer cell-specific enzyme activity include, but are not limited to, methods that utilize fluorescent probes.

[0123] Enzyme recognition sites having enzyme activity specific to cancer cells can be selected, for example, by screening fluorescent probes. In this case, when designing the enzyme recognition site, first, fluorescent probes that show a significant increase in fluorescence in tumor tissue compared to normal tissue can be selected by referring to the T / N ratio (tumor to normal ratio).

[0124] That is, another embodiment of the present invention is an "enzyme recognition site-linker binding site" represented by general formula (III), wherein the enzyme recognition site has an enzymatic activity specific to cancer cells. Y-T1 (III) (wherein Y is -OL １ (1) or -NH-L ２ (2) is selected from L １ is a partial structure of a saccharide, where the partial structure of a saccharide is a structure obtained by removing one hydroxyl group from a saccharide, and L ２ is a monovalent substituent containing an amino acid residue or an oligopeptide residue; T1 is a linker (hereinafter also referred to as "the enzyme recognition site-linker binding site of the present invention").

[0125] In the enzyme recognition site-linker binding site of the present invention, the enzyme recognition site preferably has a structure selected from the following, which was found by the above-mentioned screening.

[0126] Furthermore, more preferred enzyme recognition sites in the present invention have a structure selected from the following:

[0127] Furthermore, the linker portion in the "enzyme recognition site-linker binding site" of the present invention is selected from the group consisting of the following structures (a1) to (f1) and combinations of two or more of these:

[0128] In the above formulas (a1), (d1) to (f1), R １ ~R ４ may be the same or different, and each independently represents hydrogen, a methyl group, a methoxy group, —COOMe (Me represents a methyl group), —NHMe, F, Cl, Br, and NO ２ is selected from the group consisting of:

[0129] R in formula (e1) ５ is hydrogen or an alkyl group having 1 to 3 carbon atoms. ６ is independently in each occurrence hydrogen or an alkyl group having 1 to 3 carbon atoms.

[0130] For example, non-limiting examples of linkers having the structure (a1) include the following:

[0131] In addition, R in the formulas (d1) to (f1) １ ~R ４ At least one of may be a linking group selected from the following: Here, in the linkers represented by the formulae (d1) to (f1), R １ ~R ４ When at least one of is a linking group, the linker can be connected to X １ can be combined with

[0132] In formulas (a1) to (f1), * indicates the direction of binding to a ligand selected from an anticancer drug, an antibody, or a small molecule, a peptide, an aptamer, or a hormone-drug conjugate. Here, the linkers in formulas (a1) to (f1) and the linking groups in formulas (a1') to (a3') may be bonded to a linker having a binding site that can be used as a binding point to an antibody or a ligand, in the direction indicated by *. In addition, R in formulas (d1) to (f1) １ ~R ４At least one of may be a linker having a binding site that can be used as a binding point to an antibody or a ligand. Also, when T1 has a structure that is a combination of two or more of the formulas (a1) to (f1), the * in one or more linkers other than the linker that binds to the anticancer drug, antibody, or ligand can indicate the direction of binding to the linker. Anticancer drugs include the same anticancer drugs as those detailed in the compound of the present invention.

[0133] Examples of linkers having a binding site capable of binding to an antibody or a ligand include polyethylene glycol, alkylene, peptide, and combinations thereof. Also, known linkers used in conjugates of cell-binding molecules (cell-binding ligands or receptor analogs) and cytotoxic agents, antibody-drug conjugates, etc. can be used.

[0134] Non-limiting specific structures of the linker portion in the "enzyme recognition site-linker binding site" of the present invention include the following. * indicates the direction of binding to an anticancer drug, antibody or ligand, and the linker of the above formula may be bound to a linker having a binding site that can be used as a binding point to an anticancer drug, antibody or ligand in the direction indicated by *. In the linker of the above structure, all benzene rings are unsubstituted, but any one or more benzene rings may be substituted with any of the above R １ ~R ４ In addition, a structure in which T is a combination of three or more formulae selected from the formulae (a1) to (f1) can be constructed in the same manner as the above structures (b1') to (b3').

[0135] In addition, the linker represented by T may contain, as described above, R １ ~R ４ In addition, in the case of a linker having a structure that is a combination of two or more of the formulae (a1) to (f1), the linker of the formulae (d1) to (f1) is also included. １ ~R４ At least one of them may have a linking group selected from (a1') to (a3').

[0136] These linkers can also be bound to anticancer drugs, antibodies, or ligands via the linking groups, that is, they can be bound to anticancer drugs, antibodies, or ligands. １ ~R ４ The number of linking groups capable of binding to an anticancer drug, antibody, or ligand may be 1 or more, 2 or more, or 3 or more. The position at which the linking group is introduced may be R which is ortho-position relative to the direction of binding to the enzyme recognition site (bonding arm of the benzene ring). １ , R ２ In one aspect of the present invention, in the formulas (d1) to (f1), R １ , R ２ A linking group selected from (a1') to (a3') is introduced into one or both of the above.

[0137] Further non-limiting examples of the structure of the linker portion in the "enzyme recognition site-linker binding site" of the present invention include the following: * indicates the direction of binding to an anticancer drug, antibody, or ligand, and the linker in the above formula may be bound to a linker having a binding site that can be used as a binding point to an anticancer drug, antibody, or ligand in the direction indicated by *. Here, the linker in the structure (c1') may have another linker bound to the bonding side of the benzene ring.

[0138] The "enzyme recognition site-linker binding site" of the present invention can be used, for example, in the following manner.

[0139] (1) Use of the "enzyme recognition site-linker binding site" of the present invention to activate an antibody or ligand (such as a small molecule, peptide, aptamer, or hormone-drug conjugate) having a payload that is released upon cleavage of the binding site by an enzyme activity specific to the binding site.

[0140] (2) Use of the "enzyme recognition site-linker bond" of the present invention to prevent activation of a prodrug or binding of an antibody or ligand until the bond is cleaved by an enzyme activity specific for the bond.

[0141] (3) Use of the "enzyme recognition site-linker binding site" of the present invention to activate an antibody or ligand having a payload that is released or activated by cleavage of the binding site by an enzyme activity specific to the binding site in target cells or tissues that overexpress an oncogene, where the oncogene can be any mutated or activated gene known to drive cancer, such as the RAS or MYC oncogenes.

[0142] (4) Use of the "enzyme recognition site-linker binding site" of the present invention to target cancer, such as breast cancer, esophageal cancer, lung cancer, stomach cancer, colon cancer, small intestine cancer, pancreatic cancer, liver cancer, head and neck cancer, oral cancer, ovarian cancer, brain tumor, kidney cancer, prostate cancer, and skin cancer.

[0143] The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[0144] I. Screening of glycosidase activity-detecting fluorescent probes in human lung cancer specimens and development of a new lung cancer detection probe In investigating the development of the prodrug-type anticancer drug of the present invention, we first screened glycosidase activity-detecting fluorescent probes in human lung cancer specimens. Furthermore, we evaluated the new lung cancer detection fluorescent probes discovered from the results of this screening.

[0145] Example 1 Screening of glycosidase activity-detecting fluorescent probes in human lung cancer specimens Screening for lung cancer detection was carried out using 12 types of glycosidase-reactive fluorescent probes (see FIG. 2) according to the following procedure.

[0146] The results are shown in Figure 3. Figure 3a shows the reaction scheme of the glycosidase-reactive probe. Figure 3b shows the results of an example screening using surgically resected human lung cancer (SCC) and normal tissue. The compound number of the applied probe is indicated in each well. [Fluorescent probe] = 50 μM. The scale bar is 2 cm.

[0147] Figure 3c shows the results of a comprehensive analysis based on fluorescence imaging of glycosidase activity in normal lung, Adenocarcinoma, and SCC tissues using 12 fluorescent probes. The increase in fluorescence represents the increase from 1 to 30 minutes after addition of the fluorescent probe. Each dot represents the increase in fluorescence in normal lung, Adenocarcinoma, and SCC tissues, respectively, from left to right. Figure 3c shows that glycosidase-reactive fluorescent probes 1, 2, 5, and 11 showed an increase in fluorescence in both Ad (lung adenocarcinoma) and SCC (lung squamous cell carcinoma) tissues.

[0148] Panel d of Figure 3 shows the time-dependent fluorescence increase of probes 1 (HMRef-βGlc; also abbreviated as β-D-Glc), 2 (HMRef-βGal; also abbreviated as β-D-Gal), and 11 (HMRef-βGlcNAc; also abbreviated as β-D-GlcNAc) in normal and cancerous (Ad and SCC) lung tissues. The dotted line represents the increase in fluorescence in normal lung tissue. The black line represents the increase in fluorescence in cancerous (Ad and SCC) lung tissue. The error bars represent the standard deviation.

[0149] Panel e of Figure 3 shows the increase in fluorescence after 30 minutes in lung cancer (SCC) tissues in the presence and absence of each inhibitor. The black bars in the right panel represent the increase in fluorescence in the absence of an inhibitor, while the gray bars represent the increase in fluorescence in the presence of an inhibitor. The concentrations of the probes and inhibitors are as follows: [Fluorescent Probe] = 50 μM, [Isofagomine] = 500 μM (for Probe 1 (β-D-Glc)), [N-(n-Nonyl)deoxygalactonoijirimycin] = 500 μM (for Probe 2 (β-D-Gal)), [Swainsonine] = 500 μM (for Probe 5 (α-D-Man)), and [PUGNAc] = 500 μM (for Probe 11 (β-D-GlcNAc)). Panel f of Figure 3 shows the ROC curve of the hit probes for lung cancer detection.

[0150] The lung cancer detection ability of each probe is shown in Table 1. The threshold, sensitivity, and specificity of each probe were evaluated using the receiver operating characteristic curve. AUC is the area under the curve.

[0151]

[0152] Table 1 shows that probe 11 (HMRef-βGlcNAc) is excellent in both sensitivity and specificity. Therefore, probe 11 is highly effective as a lung cancer detection probe, which is a novel finding in light of the inventors' previous research.

[0153] Example 2 Identification of the Target Enzyme of HMRef-βGlcNAc Next, the target glycosidase of probe 11 (HMRef-βGlcNAc), whose usefulness as a lung cancer detection probe was found in Example 1, was identified as follows. The fluorescence of HMRef-βGal increased with the addition of β-galactosidase 1 (GLB1), and the fluorescence was suppressed by the addition of the inhibitor N-(n-nonyl)deoxygalactonoijirimycin. Since only GLB1 has been reported as a human galactosidase, the target enzyme of HMRef-βGal was confirmed to be GLB1. The fluorescence of HMRef-βGlcNAc increased with the addition of β-hexosaminidase A (HEXA) and β-hexosaminidase B (HEXB), and the fluorescence was suppressed by the addition of the inhibitor PUGNAc. From these results, it was presumed that the target enzymes of HMRef-βGlcNAc are HEXA and HEXB. In the DEG assay, lysates from clinical lung cancer specimens were separated by two-dimensional electrophoresis based on molecular weight and isoelectric point. When HMRef-βGlcNAc was added to the gel, a single fluorescent spot was detected. Peptide mass fingerprinting analysis of this spot identified HEXA and HEXB, demonstrating that these are the target enzymes of HMRef-βGlcNAc in lung cancer.

[0154] The specific procedure is as follows: (1) Confirmation of reactivity with the target purified protein. (1) The purified protein was added at a concentration of 10 ng / μL to a 1 μM PBS solution of each fluorescent probe, and the increase in fluorescence over 60 minutes was evaluated. Each inhibitor was added at a concentration of 10 μM.

[0155] (2) DEG Assay (1) Lysates from lung cancer clinical specimens were separated by two-dimensional electrophoresis based on molecular weight and isoelectric point, and a PBS solution containing 1 μM HMRef-βGlcNAc was added to the gel. (2) Fluorescence was measured 24 hours after addition, and fluorescent spots were excised, washed with PBS, and frozen at -70°C until evaluation. (3) The excised gel pieces were analyzed by peptide mass fingerprinting, and the target enzyme was identified from a list of identified proteins.

[0156] The results are shown in Figure 4. Figure 4(a) shows the results of fluorometric assays of hit fluorescent probes using the corresponding target purified protein in the presence and absence of each inhibitor. [Fluorescent probe] = 1 μM, [inhibitor] = 10 μM, [purified protein] = 10 ng / μL. Figure 4(b) shows the results of DEG assays of surgically resected lung Ad tissue using HMRef-βGlcNAc. A single fluorescent spot was detected. Figure 4(c) shows examples of IHC staining of GLB1, HEXA, or HEXB in normal lung, Ad, SCC, LCLC, and SCLC tissues. Malignant tissues (lung adenocarcinoma, lung squamous cell carcinoma, lung large cell carcinoma, and lung small cell carcinoma) tend to have high expression of GLB1, HEXA, and HEXB. The scale bar is 100 μm.

[0157] Example 3 Imaging of Human Lung Cancer with HMRef-βGlcNAc Next, fluorescent imaging of human lung cancer was performed using probe 11 (HMRef-βGlcNAc) according to the following procedure. As a result, it was possible to visualize lung adenocarcinoma and squamous cell carcinoma down to minute cancer tissues of 0.2 mm in size. Furthermore, similar visualization was also possible in specimens of breast cancer lung metastasis.

[0158] The specific procedure is as follows: (1) 50 μM HMRef-βGlcNAc dissolved in PBS (1-2 mL) was sprayed onto the specimen to be evaluated, and the resulting fluorescent image was evaluated using a fluorescent imaging device. (2) After fluorescent imaging, the specimen was fixed with formalin and then subjected to HE and IHC analysis and pathological diagnosis.

[0159] The results are shown in Figure 5. Application of HEXA- and HEXB-Reactive Fluorescent Probes to Lung Cancer-Specific Imaging Figure 5(a) shows time-dependent fluorescence images of a surgically resected fresh lung specimen containing both normal and cancerous (Ad) tissue after administration of HMRef-βGlcNAc. [Fluorescent Probe] = 50 μM. Figure 5(b) shows time-dependent fluorescence images of a surgically resected fresh lung specimen containing both normal and cancerous (metastatic breast cancer to the lung) tissue after administration of HMRef-βGlcNAc. [Fluorescent Probe] = 50 μM. Figure 5(c) shows time-dependent fluorescence images of a surgically resected fresh lung specimen containing both normal and cancerous (Ad) tissue after administration of HMRef-βGlcNAc. [Fluorescent Probe] = 50 μM. Figure 5(d) shows a magnified fluorescence image at 30 minutes. Figure 5(e) shows the increase in fluorescence and pathology for each ROI shown in figure 5(d). Panel f of Figure 5 shows the results of histological and IHC analyses of each target glycosidase (HEXA or HEXB). In cases 1-3, the pathological cancer lesions are surrounded by dotted lines.

[0160] Example 4: Example of cancer visualization following intraperitoneal and systemic administration of HMRef-βGlcNAc. Probe 11 (HMRef-βGlcNAc) was used to visualize cancer following intraperitoneal and systemic administration according to the following procedure. HMRef-βGlcNAc was capable of clearly imaging even minute cancer lesions of 1 mm or less in peritoneal dissemination. Furthermore, intravenous administration also clearly visualized the lung cancer site in an orthotopic lung cancer transplant model, and background fluorescence in organs other than the cancer site was significantly suppressed. These results demonstrate that HMRef-βGlcNAc is useful for cancer visualization, even when administered intraperitoneally or systemically.

[0161] The specific procedure is as follows: (1) Study on intraperitoneal administration (1) BALB / cAJcl-nu / nu (7 weeks old) was injected with 5 x 10 luciferase-expressing A549 cells. ６(2) 50 μM HMRef-βGlcNAc dissolved in PBS (300 μL) was administered intraperitoneally to the peritoneal dissemination model mice. (3) After one hour, the mice were sacrificed by cervical dislocation, and the abdomen was opened to remove the intestine and mesentery. Fluorescence imaging was performed using an in vivo fluorescence imaging system.

[0162] (2) Study on intravenous administration (1) Model mice were prepared using BALB / cAJcl-nu / nu (7 weeks old) as follows: The skin of the chest was incised, the subcutaneous tissue was removed, and 2 × 10 luciferase-expressing A549 cells were injected into the left lung through the intercostal pleura. ７ 100 cells / mL (50 μL, 5% Matrigel) was injected into the mice. After injection, the skin was sutured. The tumor was allowed to grow for one month to create a lung cancer orthotopic transplant mouse model. (2) 10 mg / kg HMRef-βGlcNAc dissolved in PBS (100 μL) was administered intravenously to the lung cancer orthotopic transplant mice. (3) After 30 minutes, the mice were sacrificed by cervical dislocation, and then the abdomen and thoracotomy were performed to expose the lungs, and ex vivo fluorescent imaging was performed using a fluorescent imaging device.

[0163] The results are shown in Figure 6. Panel (a) of Figure 6 shows the results of ex vivo fluorescence imaging of freshly resected peritoneal cavities after intraperitoneal administration of 50 μM HMRef-βGlcNAc to A549 peritoneal metastasis mice. Panel (b) of Figure 6 shows the results of ex vivo fluorescence imaging of freshly resected lungs after intravenous administration of 10 mg / kg HMRef-βGlcNAc to A549 lung cancer orthotopically transplanted mice.

[0164] II. Development of Prodrug-Type Anticancer Agents of the Present Invention Next, the development of novel prodrug-type anticancer agents was investigated by introducing into the SN38 molecule a substrate moiety that targets the cancer-specific enzyme activity discovered by screening of the fluorescent probe in human lung cancer specimens in Example 1.

[0165] (A) Development of Novel Prodrug-Type Anticancer Agents Targeting Glycosidase Activity 1. Synthesis of Prodrug Compounds Among the substrate moieties confirmed in Example 1 to be promising as substrate moieties targeting cancer-specific enzyme activity, -βGal, -βGlcNAc, and -αMan were introduced into the SN38 molecule to synthesize three types of SN38-based prodrug compounds by the following procedure.

[0166] Synthesis Example 1 Synthesis of SN38-βGal SN38-βGal, a compound of the present invention, was synthesized according to the following scheme 1.

[0167] Scheme 1

[0168] 7-Ethyl-10-hydroxycaptothecin (SN38) (190 mg, 0.484 mmol), 2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl bromide (1.50 g, 3.65 mmol), Cs ２ CO ３ (1.00 g, 3.07 mmol) was dissolved in 10.0 mL of acetonitrile and stirred at room temperature for 3 hours. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (CH ２ Cl ２ An acetylated glycoadduct of SN38 was obtained by distilling off 5.0 mL of MeOH (MeOH = 95:5). This was dissolved in 5.0 mL of MeOH, and NaOMe (130 mg, 2.41 mmol) dissolved in 5.0 mL of MeOH was added dropwise, followed by stirring at room temperature for 1 hour. The solvent was distilled off under reduced pressure, and the resulting mixture was purified by HPLC and then lyophilized to obtain the target product SN38-βGal (188 mg, 0.339 mmol) as a yellow solid in a yield of 70.1%. The target product obtained was １ H NMR, １３ The product was identified by CNMR and ESI-HRMS.

[0169] 1H NMR (400 MHz, DMSO-d6): δ8.10 (d, J = 9.6 Hz, 1H), 7.74 (s, 1H), 7.58 (dd, J = 1.2 Hz, 9.2 Hz, 1H), 7.28 (s, 1H), 5.43 (s, 2H), 5.30 (s, 2H), 5.10 (d, J = 7.6 Hz, 1H, overlaps with HOD), 3.75-3.68 (m, 3H), 3.60-3.20 (m, 2H), 3.50 (dd, J = 2.4 Hz, 9.2 Hz, 1H), 3.21-3.20 (m, 2H), 1.95-1.80 (m, 2H), 1.31 (t, J = 7.2 Hz, 3H), 0.88 (t, J = 7.2 Hz, 3H). 13 C NMR (101 MHz, DMSO-d6): δ172.5, 156.8, 156.1, 150.1, 150.0, 146.2, 144.7, 144.2, 131.2, 128.3, 127.6, 122.5, 118.4, 106.6, 101.1, 96.1, 75.9, 73.3, 72.4, 70.2, 68.2, 65.2, 60.5, 49.5, 30.2, 22.2, 13.5, 7.7. ESI-HRMS (ESI +) m / z calcd. for [M+Na]+, 577.17981; found, 577.18002.

[0170] Synthesis Example 2 Synthesis of SN38-βGlcNAc SN38-βGlcNAc, a compound of the present invention, was synthesized according to the following Scheme 2.

[0171] Scheme 2

[0172] 7-Ethyl-1-10-hydroxycamptothecin (SN38) (1.12 g, 2.85 mmol), 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-D-glucopyranosyl chloride (3.2 g, 8.75 mmol), Cs ２ CO ３(3.1 g, 9.51 mmol) was dissolved in 100 mL of acetonitrile and stirred at room temperature for 3 hours. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (CH ２ Cl ２ An acetylated glycoadduct of SN38 was obtained by distilling off 5.0 mL of NaOMe (700 mg, 13.0 mmol) dissolved in 5.0 mL of MeOH (95:5). This was dissolved in 30 mL of MeOH, and NaOMe (700 mg, 13.0 mmol) dissolved in 5.0 mL of MeOH was added dropwise, followed by stirring at room temperature for 1 hour. The solvent was distilled off under reduced pressure, and the resulting mixture was purified by HPLC and lyophilized to obtain the target product SN38-βGlcNAc (456 mg, 0.766 mmol) as a yellow solid in a yield of 26.8%. The target product obtained was １ H NMR, １３ The product was identified by CNMR and ESI-HRMS.

[0173] 1 H NMR (400 MHz, CD3OD): δ7.57 (d, J = 9.2 Hz, 1H), 7.38 (s, 1H), 7.24 (s, 1H), 7.20 (d, J = 2.4 Hz, 1H), 5.39 (d, J = 16 Hz, 1H), 5.17 (d, J = 16 Hz, 1H), 5.13 (d, J = 8.4 Hz, 1H), 3.99-3.93 (m, 2H), 3.75 (dd, J = 6.8 Hz, 12 Hz, 1H), 3.62 (t, J = 8.8 Hz, 1H), 3.55-3.51 (m, 1H), 3.39 (t, J = 9.2 Hz, 1H), 2.98-2.94 (m, 2H), 1.98 (s, 3H), 1.86-1.78 (m, 2H), 1.26 (t, J = 7.6 Hz, 3H), 0.90 (t, J = 7.6 Hz, 3H). 13C NMR (101 MHz, CD3OD): δ174.8, 174.0, 158.8, 157.7, 152.6, 160.6, 147.4, 146.5, 145.8, 131.8, 129.0, 128.7, 123.6, 119.8, 108.1, 100.8, 99.1, 79.0, 75.8, 74.1, 72.2, 66.6, 62.8, 57.4, 40.4, 32.2, 23.9, 23.2, 14.1, 8.2. ESI-HRMS (ESI +) m / z calcd. for [M+Na]+, 618.20582; found, 618.20633.

[0174] Synthesis Example 3 Synthesis of SN38-αMan SN38-αMan, a compound of the present invention, was synthesized according to the following scheme 3.

[0175] Scheme 3

[0176] 7-Ethyl-10-hydroxycamptothecin (SN38) (100 mg, 0.255 mmol) and 1,2,3,4,6-penta-O-acetyl-D-mannopyranose (1.00 g, 2.56 mmol) were dissolved in 8.0 mL of dichloromethane, and BF ３ ・Et ２ After adding 2.5 mL of pyridine (2.5 mL) and stirring at room temperature under an argon atmosphere for 48 hours, the organic phase was neutralized with saturated aqueous sodium bicarbonate, washed with water and saturated brine, and dehydrated with sodium sulfate. After removing the sodium sulfate, the solvent was evaporated under reduced pressure and purified by silica gel column chromatography (CH ２ Cl ２ An acetylated sugar adduct of SN38 was obtained by distilling off SN38 in 4.0 mL of MeOH (MeOH = 95:5). This was dissolved in 4.0 mL of MeOH, and NaOMe (150 mg, 2.78 mmol) dissolved in 4.0 mL of MeOH was added dropwise, followed by stirring at room temperature for 1 hour. The solvent was distilled off under reduced pressure, and the resulting mixture was purified by HPLC and then lyophilized to obtain the target product SN38-αMan (49.0 mg, 0.0884 mmol) as a yellow solid in a yield of 34.7%. The target product obtained was １ H NMR, １３ The product was identified by CNMR and ESI-HRMS.

[0177] 1 H NMR (400 MHz、 DMSO-d6): δ8.03 (dd、 J = 1.2 Hz、 9.2 Hz、 1H)、 7.76 (s、 1H)、 7.54 (d、 J = 9.2 Hz、 1H)、 7.21 (d、 J = 1.2 Hz、 1H)、 5.58 (s、 1H)、 5.36 (s、 2H)、 5.23 (s、 2H)、 3.87 (d、 J = 1.6 Hz、 1H)、 3.70 (dd、 J = 2.4 Hz、 9.2 Hz、1H)、 3.57-3.38 (m、 4H)、 3.09 (d、 J = 7.6 Hz、 2H)、1.90-1.70 (m, 2H)、 1.25 (t、 J = 7.6 Hz、 3H)、 0.81 (t、 J = 5.2 Hz、 3H). 13 C NMR (101 MHz、 DMSO-d6): δ172.5、 156.8、 155.1、 150.1、 150.0、 146.2、 144.8、 144.2、 131.3、 128.3、 127.6、 122.9、 118.4、 107.2、 99.1、 96.1、 75.1、 72.4、 70.6、 70.0、 66.7、 65.2、 61.0、 49.5、 30.2、 22.3、 13.5、 7.7. ESI-HRMS (ESI +) m / z calcd. for [M+Na]+、 577.17927; found、 577.17869.

[0178] Example 5: Cytotoxicity Test of Developed Prodrugs Using the MTT Assay A cytotoxicity test of SN38-βGal and SN38-βGlcNAc was performed on lung adenocarcinoma A549 cells using the MTT assay. As a result, cell proliferation was inhibited with increasing concentrations of SN38-βGal and SN38-βGlcNAc (Figure 7). Under the following measurement conditions, the calculated IC50 values ​​were 154 nM for SN38, 3.38 μM for SN38-βGal, 1.35 μM for SN38-βGlcNAc, and 30.7 μM for CPT-11. SN38-βGal and SN38-βGlcNAc showed lower IC50 values ​​than CPT-11. Furthermore, it was confirmed that the cytotoxicity of SN38-βGlcNAc was significantly reduced by the addition of PUGNAc, an inhibitor of the target enzyme, β-hexosaminidase. In this test, Cell Proliferation Kit I (MTT), SIGMA-ALDRICH, was used.

[0179] The specific procedure for the cytotoxicity test is as follows: (1) In a 96-well plate, 1.0 x 10 A549 cells were placed. ４ 100 μL of DMEM containing cells, 10% FBS, and 1% PS was added, and the cells were incubated at 37°C and 5% CO ２ (2) 10 μL of medium containing each drug concentration was added, and the cells were incubated at 37°C, 5% CO ２ (3) 10 μL of MTT labeling reagent was added, and the mixture was incubated at 37°C and 5% CO for 4 hours. ２ (4) 100 μL of solubilization buffer was added, and the mixture was incubated in a 5% CO ２ The cells were incubated in an ambient atmosphere for 24 hours. (5) The absorbance at 560 nm was measured to evaluate cell viability, and the IC50 value was calculated.

[0180] Figure 7 shows the results of the cytotoxicity test by MTT assay. Panel a of Figure 7 shows the results of cell growth inhibition of lung adenocarcinoma A549 cells at each drug concentration, and panel b of Figure 7 shows the cell growth inhibition curve and IC50 value of each drug. Panel c of Figure 7 shows the suppression of drug efficacy in the presence of an inhibitor.

[0181] Example 6 Cytotoxicity Test Using JFCR39 Cell Panel The cell proliferation of SN38-βGal, SN38-βGlcNAc, SN38, and CPT-11 was evaluated for a total of 39 cell lines, including the JFCR39 cell panel (7 lung cancer lines, 6 gastric cancer lines, 5 colon cancer lines, 5 ovarian cancer lines, 6 brain tumor lines, 5 breast cancer lines, 2 renal cancer lines, 2 prostate cancer lines, and 1 melanoma line). As shown in Figures 8 to 11, SN38-βGal and SN38-βGlcNAc inhibited the proliferation of almost all cancer cells, and showed GI50 values ​​similar to those of SN38 but lower than those of CPT-11 (Table 2).

[0182] The specific test procedure is as follows: Cancer cells were plated on a 96-well plate, and the next day, a drug solution was added in a dilution series. After culturing for 2 days (48 hours), cell proliferation was measured by colorimetry using sulforhodamine B.

[0183]

[0184] Example 7: Evaluation of drug release using LC-MS. LC-MS was used to confirm the reactivity of SN38-βGal with human β-galactosidase 1 (GLB1), the presumed target enzyme. The release of SN38, which exhibits anticancer activity, was confirmed upon addition of purified GLB1 enzyme (Figure 12).

[0185] 12 shows the results of LC-MS analysis confirming the reactivity of SN38-βGal with GLB1. SN38-βGal releases SN38, which has anti-cancer activity, in the presence of GLB1.

[0186] Example 8: Kinetics study of intraperitoneal administration (ip) of SN38-βGal in mice (intraperitoneal). Pharmacokinetics was evaluated following intraperitoneal administration (ip) of 30 mg / kg SN38-βGal. The blood concentration of SN38-βGal was highest approximately 10 minutes after administration and had almost completely disappeared by 60 minutes. The blood half-life was approximately 30 minutes. Evaluation of intraperitoneal concentration changes showed that the intraperitoneal concentration decreased to approximately 17% by 30 minutes and had almost completely disappeared from the peritoneal cavity by 60 minutes. Organ distribution of SN38-βGal was evaluated, and distribution in each organ was confirmed between 10 and 60 minutes after administration. Absorption from the small intestine was particularly significant, with only a slight distribution observed in the brain. Small amounts of the active form of SN38 were also detected in each organ, with significant concentrations observed in the small intestine and large intestine (Figure 13). The pharmacokinetic parameters of SN38-βGal following intraperitoneal administration are shown in Table 3.

[0187] The specific procedure for the pharmacokinetics study is as follows: (1) SN38-βGal (30 mg / kg) was dissolved in 100 μL of PBS and administered i.p. (2) 150-200 μL of anesthesia (in the case of a triple combination of Domitor, Betolfar, and Midazolam) was administered intraperitoneally 5 minutes before dissection at each time point. (3) The inside of the syringe was coated with heparin sodium, and at each evaluation time point, the mouse was quickly placed on a dissection pad. The abdomen was opened to prevent bleeding, and whole blood was collected from the right ventricle using a 23G needle. 100 μM β-galactosidase inhibitor was added to the whole blood. (4) After whole blood collection, the brain, heart, lungs, liver, pancreas, spleen, kidneys, stomach, small intestine, large intestine, muscle, and fat were removed. Muscle was excised from the thigh area. Each organ was weighed. (5) Whole blood was centrifuged at 1200G for 15 minutes at 4°C, and 200 μL of the supernatant was collected. 800 μL of a 1:1 MeOH / MeCN solution (containing 25 μM Rhodamine 123 as an internal standard) was added and allowed to stand for 10 minutes. (6) After standing, the mixture was centrifuged at 1700G for 15 minutes at 4°C, and the supernatant was collected. Approximately 80 μL of the mixture was taken and loaded onto a Cosmos Spin Filter G (0.2 μm). The filtered sample was collected by centrifugation at 12000G for 5 minutes at 4°C. (7) Each excised organ was finely minced and placed in a Lysing Matrix D tube. 1000 μL of Lysis Buffer (containing 100 μM β-galactosidase inhibitor) was added, and the organs were disrupted using a shaker. (8) The supernatant was collected from the Lysing Matrix tube and centrifuged at 10,000 G for 5 minutes at 4°C to obtain the supernatant. (9) 200 μL of MeOH / MeCN (1:1, containing 30 μM Rhodamine 123 as an internal standard) was added to 100 μL of the organ lysate solution and allowed to stand for 10 minutes. (10) 200 μL of the supernatant was collected by centrifugation at 1,700 G for 15 minutes at 4°C, loaded onto a Cosmos Spin Filter G (0.2 μm), and centrifuged at 12,000 G for 5 minutes at 4°C to collect the sample.(11) 50 μL of purified water was mixed with 50 μL of filtered plasma sample or organ lysate sample, and 10 μL of the mixture was injected into HPLC. Data on the area under the curve of the PDA spectrum at 380 nm (derived from SN38) and 490 nm (derived from Rhodamine 123) were obtained, and then the values ​​for blood were calculated from the volume and for organs from the weight using a single calibration point for Rhodamine 123.

[0188] The specific procedure for the intraperitoneal concentration evaluation test is as follows: (1) The evaluation dose of drug was administered intraperitoneally (i.p.). (2) At each evaluation time, 2.0 mL of PBS was administered i.p., and after administration, the abdomen was quickly massaged and sacrificed to recover as much drug as possible into the PBS. A small hole was made in the abdomen, and the PBS in the peritoneal cavity was quickly recovered with a 1.0 mL syringe by pushing it out. This solution was used as peritoneal lavage fluid. (3) To 200 μL of the recovered peritoneal lavage fluid, 800 μL of 1:1 MeCN / MeOH (containing 25 μM Rhodamine 123) was added to precipitate proteins. The supernatant was recovered by centrifugation at 1700×G for 5 minutes at 4°C. (4) 80 μL of the recovered supernatant was loaded onto a Cosmos Spin Filter G and centrifuged at 12,000 G for 5 minutes at 4°C. 50 μL of the recovered material was mixed with 50 μL of purified water to make a total volume of 100 μL, and the sample was prepared. (5) The concentration of SN38-βGal was measured by HPLC, and the concentration of the recovered peritoneal lavage fluid was calculated.

[0189]

[0190] Figure 13 shows the results of pharmacokinetics following intraperitoneal administration (i.p.) of 30 mg / kg of SN38-βGal. Panel a of Figure 13 shows the change in blood concentration. Panel b of Figure 13 shows the change in intraperitoneal concentration, and panel c of Figure 13 shows the organ distribution of SN38-βGal and SN38.

[0191] Example 9 Toxicity Test (1) Single-Dose Toxicity Test 1 Mice (Intravenous) Single-dose toxicity was evaluated by intravenously administering 200 mg / kg each of the novel prodrug anticancer agents SN38-βGal and SN38-βGlcNAc to Jcl:ICR mice (female). Immediately after administration, decreased spontaneous activity, tremors, and prone positioning were observed in more than half of the mice in both groups, but these symptoms improved within several hours after administration. In both groups, no deaths were observed immediately after administration or 10 days later, so the LD50 value was estimated to be 200 mg / kg or higher (Table 4). The LD50 value of the existing drug CPT-11 has been reported to be 132.4 mg / kg in mice (female), and it was confirmed that the LD50 value was significantly higher than that of CPT-11.

[0192]

[0193] (2) Single-Dose Toxicity Test 2: Mice (Intravenous) The novel prodrug anticancer agents SN38-βGal and SN38-βGlcNAc were intravenously administered to Jcl:ICR mice (female) to evaluate single-dose toxicity. SN38-βGal was intravenously administered at 300 mg / kg, due to its upper limit of solubility. SN38-βGlcNAc was intravenously administered at 500 mg / kg. Immediately after administration, more than half of the mice in both groups showed decreased locomotor activity and prone positioning, but these symptoms improved within several hours after administration. Since no deaths were observed immediately or 14 days after administration in either group, the LD50 values ​​were estimated to be 300 mg / kg or higher for SN38-βGal and 500 mg / kg or higher for SN38-βGlcNAc (Table 5). The LD50 value of the existing drug CPT-11 has been reported to be 132.4 mg / kg in mice (female), so it was confirmed that the LD50 value was significantly higher than that of CPT-11.

[0194] The specific toxicity test procedure is as follows: (1) Jcl:ICR mice (7-week-old, female) were intravenously administered 100 μL of the drug dissolved in PBS via the tail vein. (2) The condition immediately after administration was observed for approximately 30 minutes, and the survival rate 14 days after administration was evaluated.

[0195]

[0196] (3) Repeated-Dose Toxicity Test in Mice (Intravenous) Toxicity evaluation was performed on Jcl:ICR mice (female) by repeated administration of 40 mg / kg of SN38-βGal, SN38-βGlcNAc, and CPT-11. Each dose was administered intravenously (i.v.) three times a week for three weeks, and weight changes were observed. The vehicle group was administered the corresponding amount of DMSO. As a result, a maximum weight loss of approximately 20% was observed in multiple mice in the CPT-11-administered group by the final day. On the other hand, the SN38-βGal and SN38-βGlcNAc-administered groups did not exhibit such significant weight loss, and almost no difference in weight change was observed compared to the vehicle group (Figure 14).

[0197] Figure 14 shows the changes in body weight over time following repeated administration of the novel prodrug and CPT-11. Figure 14 confirms a tendency for weight loss with the existing drug CPT-11 compared to SN38-βGal and SN38-βGlcNAc. The black arrow indicates the day of i.v. administration of 40 mg / kg. n=8.

[0198] Furthermore, symptoms such as decreased spontaneous activity, tremors, prone position, and exophthalmos were observed immediately after administration of CPT-11. On the other hand, no symptoms were observed immediately after administration of SN38-βGal or SN38-βGlcNAc. After blood collection on day 22, the animals were euthanized, and the liver, kidneys, and spleen were removed and compared in weight. A slight decrease in kidney weight was observed in the CPT-11-administered group, but this tendency was not observed in the SN38-βGal or SN38-βGlcNAc-administered group. A tendency for spleen weight to increase was observed in the SN38-βGlcNAc and CPT-11-administered groups, and this tendency was particularly pronounced in the CPT-11-administered group ( FIG. 15 ).

[0199] The collected organs were subjected to histological analysis (FIGS. 16 to 20), and blood tests were performed on the whole blood and serum. The main findings obtained in comparison with the vehicle group are shown below (Tables 6 to 8).

[0200] SN38-βGal Biochemical testing showed slight decreases in AST and ALT levels. Blood cell testing suggested a slight increase in eosinophils and a decrease in basophils. Histological findings showed no organ damage in the small intestine, large intestine, kidneys, liver, or spleen.

[0201] SN38-βGlcNAc An increase in spleen weight was observed. Biochemical tests showed slight increases in urea nitrogen and creatinine. Hematology showed an estimated increase in eosinophils and a decrease in basophils. Histological findings showed no organ damage in the small intestine, large intestine, kidneys, liver, or spleen.

[0202] CPT-11: Weight loss of up to approximately 20% was observed. Decreases in liver and kidney weight and a clear increase in spleen weight were observed. Biochemical tests showed decreases in albumin, A / G ratio, AST, ALT, and ALP, and increases in urea nitrogen and creatinine. In addition, increases in the proportion of neutrophils and monocytes, and decreases in the proportion of lymphocytes and basophils were observed. Hematological test values ​​indicated side effects mainly on the kidneys, liver, and gastrointestinal tract, and these side effects were considered to be more significant than those of SN38-βGal and SN38-βGlcNAc. Histological findings showed severe organ damage in the small intestine and large intestine. Organ damage in the kidneys, liver, and spleen was very mild or absent.

[0203] From the above results, it was estimated that the toxicity of the novel prodrugs SN38-βGal and SN38-βGlcNAc was extremely mild compared to CPT-11, based on a comprehensive perspective including changes in body weight, blood tests, changes in organ weight, histological analysis, and post-administration conditions, and that no serious organ damage would occur under the administration period and conditions of this study.

[0204] The histological findings are shown below.

[0205] Histological findings in the small intestine: In the CPT-11 group, (1) erosion, (2) loss of germ cells, (3) atrophy, loss, and degeneration of glandular ducts, (4) interstitial fibrosis, (5) inflammatory cell infiltration of lymphocytes, and (6) degeneration and widespread necrosis of the lamina propria of the mucosa were observed, indicating serious organ damage. In contrast, the SN38-βGal and SN38-βGlcNAc groups showed only slight erosion, (2) slight loss of germ cells, (3) slight ductal degeneration, and (5) mild lymphocyte infiltration, with almost no interstitial fibrosis, indicating extremely mild side effects compared to CPT-11. (Figure 16)

[0206] Histological findings in the large intestine: In the CPT-11-treated group, (1) erosion, (2) loss of germ cells, (3) atrophy, loss, and degeneration of glandular ducts, (4) interstitial fibrosis, and (5) inflammatory cell infiltration of lymphocytes were observed, indicating serious organ damage. In contrast, in the SN38-βGal and SN38-βGlcNAc-treated groups, only (1) slight erosion, (2) slight loss of germ cells, (3) slight degeneration of glandular ducts, and (5) mild lymphocyte infiltration were observed, with almost no interstitial fibrosis or other side effects, indicating extremely milder side effects than those of CPT-11 (Figure 17).

[0207] Histological findings in the kidneys: In the CPT-11-administered group, (1) slight lymphocytic infiltration and (2) tubular loss (indicated by the black arrow) were observed in the kidneys, but no significant changes were observed in the organ as a whole, and organ damage to the kidneys was extremely mild. In the SN38-βGal and SN38-βGlcNAc-administered groups, slight lymphocytic infiltration was also observed, but no significant changes were observed in the organ as a whole, and no organ damage to the kidneys was observed in the novel prodrug groups either (Figure 18).

[0208] Histological findings in the liver In the CPT-11-administered group, (1) hepatocyte nuclear enlargement (increased chromatin) was frequently observed, and (2) lymphocyte infiltration was slightly observed, but overall liver damage was mild. In the SN38-βGal and SN38-βGlcNAc-administered groups, (1) hepatocyte nuclear enlargement (normal chromatin) was also slightly observed, but no major changes were observed overall in the organ, and no liver damage was observed in the novel prodrug groups (Figure 19).

[0209] Histological findings in the spleen In the spleen, there was a tendency for some multinucleated cells and splenomegaly to be observed in the CPT-11, SN38-βGal, and SN38-βGlcNAc-treated groups, but no major changes were observed in the organ as a whole, and organ damage to the spleen was extremely mild (Figure 20).

[0210]

[0211]

[0212]

[0213] (4) Evaluation of acetylcholinesterase (AChE) inhibitory activity The results of toxicity tests suggested that SN38-βGal and SN38-βGlcNAc hardly exhibited side effects of cholinergic syndrome (decreased locomotor activity, tremor, prone position), which is an acute toxicity that occurs in the administrable concentration range of Irinotecan (CPT-11). Since cholinergic syndrome is thought to be caused by the inhibition of AChE by CPT-11, the AChE inhibitory activity of the newly developed prodrugs SN38-βGal, SN38-βGlcNAc, and SN38-αMan was evaluated in comparison with CPT-11. As a result, in the presence of 10 μM of each drug, CPT-11 significantly inhibited human AChE activity, whereas SN38-βGal, SN38-βGlcNAc, and SN38-αMan did not inhibit AChE activity in this concentration range (Figure 21a, b). The AChE inhibitory activities of CPT-11, SN38-βGal, and SN38-βGlcNAc were examined in a concentration-dependent manner. The IC50 values ​​for AChE inhibition of each drug were calculated to be 10.6 μM for CPT-11, 1.56 mM for SN38-βGal, and 1.44 mM for SN38-βGlcNAc. SN38-βGal and SN38-βGlcNAc showed IC50 values ​​that were more than two orders of magnitude higher than that of CPT-11 (Figure 21c). These results demonstrate that the newly developed prodrugs SN38-βGal, SN38-βGlcNAc, and SN38-αMan have significantly lower AChE inhibitory activity than CPT-11, supporting the finding that no cholinergic syndrome was observed.

[0214] The specific procedure for the inhibition test is as follows: (1) Test drugs were added at various concentrations to a 96-well plate, and acetylcholine, AChE substrate, and purified AChE enzyme were added according to the protocol using the SensoLyte® 520 Acetylcholinesterase Activity Assay Kit Fluorometric (AnaSpec, Inc.). Neostigmine bromide, a known AChE inhibitor, was used as a negative control. (2) Fluorescence values ​​at 520 nm were measured, and IC50 values ​​were calculated.

[0215] 21A shows the AChE activity inhibition evaluation when 10 μM of each drug was added, and FIG. 21B shows the comparison of inhibitory activity at 30 minutes. FIG. 21C shows the concentration-dependent AChE inhibitory activity and IC50 value of each drug at 60 minutes. Error bars = SD.

[0216] Example 10: Evaluation of Locomotion After Drug Administration Locomotion after drug administration was compared (n=4). A significant decrease in locomotion was observed immediately after intravenous administration of 40 mg / kg of CPT-11. On the other hand, SN38-βGal and SN38-βGlcNAc showed almost no decrease in locomotion (Figure 22). Cholinergic syndrome has been reported as an acute toxicity after administration of CPT-11, and the decrease in locomotion is thought to be due to this. SN38-βGal and SN38-βGlcNAc did not cause cholinergic syndrome, suggesting that their acute toxicity is weaker than that of the existing drug CPT-11.

[0217] Figure 22 shows the results of the assessment of locomotion after drug administration. Figure 22a shows the head movement distance for 15 minutes from 1 minute after administration in each group. The drug was administered intravenously at 40 mg / kg (n=4). Figure 22b shows an example of the movement trajectory for 15 minutes from 1 minute after administration in each group. Figure 22c shows an example of the movement distance for each time period from 1 minute after administration in each group.

[0218] Example 11: Therapeutic Experiment in a Peritoneal Dissemination Model The therapeutic effect of SN38-βGal was verified using a peritoneal dissemination model. SN38-βGal was administered at 10 mg / kg and 20 mg / kg, and compared with a vehicle group (DMSO administration) to evaluate whether tumor suppression effects were confirmed. Each group consisted of three mice, and evaluation was performed on a BALB / cAJcl-nu / nu luciferase-expressing A549 cell peritoneal dissemination mouse model. Administration was intraperitoneal (i.p.) for 40 consecutive days. Tumor volume was assessed by evaluating luciferin / luciferase chemiluminescence. The endpoint was a significant weight loss (20% reduction) or the time point at which intraperitoneal bleeding was confirmed. Results showed that tumor growth was significantly suppressed in the administration group compared to the vehicle group. Furthermore, between the 10 mg / kg and 20 mg / kg doses, although the 20 mg / kg group showed a slightly higher tumor suppression effect, no significant difference in effect was observed. Furthermore, on the 29th day of administration, one mouse in the vehicle group reached the endpoint and died, but no deaths were confirmed in the treatment group. After the evaluation was completed, the mice were laparotomized to check for the presence or absence of tumors, and a significant reduction in peritoneal dissemination lesions was confirmed macroscopically in the treatment group compared to the vehicle. The fluorescent reagent gGlu-HMRG, which can visualize A549 tumor lesions, was sprayed, and the tumor lesions were fluorescently visualized and photographed. Significantly fluorescent areas (tumor sites) were confirmed in the vehicle, while the number of these areas was significantly reduced in the treatment group. Furthermore, when weight transition was evaluated throughout this study, slight weight loss was confirmed in the 20 mg / kg group by the 10th day of administration, but thereafter no significant weight loss was observed compared to the vehicle group. In the 10 mg / kg administration group, almost no difference was observed compared to the vehicle group. Furthermore, no noticeable side effects were observed macroscopically immediately after SN38-βGal administration. These results confirmed the effectiveness of SN38-βGal treatment in peritoneal dissemination model mice.

[0219] The specific procedure for the therapeutic experiment is as follows: (1) BALB / cAJcl-nu / nu (7 weeks old) was inoculated with 5 x 10 luciferase-expressing A549 cells. ６Cells / mL (200 μL) were administered intraperitoneally. (2) One week later, the formation of peritoneal dissemination was confirmed by luciferin / luciferase chemiluminescence, and drug administration was initiated. Luciferin was administered intraperitoneally at 3 mg (200 μL). After anesthesia with isoflurane, chemiluminescence levels were measured 20 minutes after administration using an in vivo imaging device. Drugs were prepared by dissolving 100 mM DMSO stock in PBS, and administered in a total volume of 100 μL. (3) On the day of evaluation, tumor burden was assessed by luciferin / luciferase chemiluminescence. (4) After treatment, gGlu-HMRG (10 μM, 500 μL PBS) was administered intraperitoneally. 10 minutes later, the abdomen was opened and the intestine and peritoneum were removed. (5) Fluorescence imaging of disseminated lesions was performed using an in vivo fluorescence imaging device, and the results were compared between the vehicle and administration groups.

[0220] Figure 23 shows the results of therapeutic evaluation of A549 peritoneal dissemination model mice using SN38-βGal. Panel a of Figure 23 shows the change in luminescence intensity in each group. Panel b of Figure 23 shows the time-dependent change in luminescence images in the vehicle and 20 mg / kg administration groups. Panel c of Figure 23 shows the results of confirmation of peritoneal tumor lesions after evaluation using white light images (left) and fluorescent images using gGlu-HMRG (right). Panel d of Figure 23 shows the time course of weight change in each group.

[0221] Example 12: Therapeutic Experiment 1 in an Orthotopic Lung Cancer Transplant Model Therapeutic effects of SN38-βGal, SN38-βGlcNAc, and CPT-11 were examined in an orthotopic lung cancer transplant model. The SN38-βGal, SN38-βGlcNAc, and CPT-11-administered groups were compared with a vehicle group (DMSO administration) to evaluate whether tumor-suppressing effects were confirmed. The vehicle group consisted of n=6, the SN38-βGal group n=7, the SN38-βGlcNAc group n=8, and the CPT-11 group n=7. Evaluation was performed on a BALB / cAJcl-nu / nu luciferase-expressing A549 cell peritoneal dissemination mouse model. Administration was intravenous (i.v.). The dose was 40 mg / kg, administered three times a week for four consecutive days. Tumor volume was assessed by evaluating the luciferin / luciferase chemiluminescence value. The endpoint was the time point when significant weight loss (20% reduction) occurred. As a result, compared to the vehicle group, SN38-βGal and SN38-βGlcNAc tended to suppress tumor growth, similar to the CPT-11-administered group. From day 26 onwards, when administration was terminated, tumor regrowth was observed in the SN38-βGal group (Figure 24).

[0222] Furthermore, when weight transition was evaluated throughout this study, no significant weight loss was observed in either group compared to the vehicle group. Furthermore, no significant side effects were observed macroscopically, such as immediately after administration of SN38-βGal and SN38-βGlcNAc. Meanwhile, significant weight loss was observed in the CPT-11 administration group over the first two weeks, but weight recovery was observed after two weeks. Although some weight loss was observed in the SN38-βGal and SN38-βGlcNAc administration group, it was not as significant as with CPT-11. These results demonstrate that SN38-βGal and SN38-βGlcNAc can provide treatment to the same extent or even greater extent than CPT-11 without causing weight loss.

[0223] The specific procedure for the therapeutic experiment is as follows: (1) Model mice were prepared using BALB / cAJcl-nu / nu (7 weeks old) as follows: The skin of the chest was incised, the subcutaneous tissue was removed, and 2 × 10 luciferase-expressing A549 cells were injected into the left lung through the intercostal pleura. ７ Cells / mL (50 μL, 5% Matrigel) were injected. After injection, the skin was sutured. (2) After 17 days, tumor formation was confirmed by luciferin / luciferase chemiluminescence, and intravenous drug administration was initiated. Drugs were prepared by dissolving 200 mM DMSO stock in PBS, and administered in a total volume of 100 μL. Luciferin was administered intraperitoneally at 3 mg (200 μL). After anesthesia with isoflurane, chemiluminescence levels were measured 20 minutes after administration using an in vivo imaging device. (3) On the day of evaluation, tumor burden was assessed by body weight and luciferin / luciferase chemiluminescence.

[0224] Figure 24 shows the results of therapeutic evaluation of SN38-βGal, SN38-βGlcNAc, and CPT-11 in an A549 lung cancer orthotopic transplant model. Panel (a) of Figure 24 shows the dosing schedule, and panel (b) of Figure 24 shows the tumor suppression effect in each administration group. Panel (c) of Figure 24 shows the change in body weight over the treatment period in each group.

[0225] Example 13: Therapeutic Experiment 2 in an Orthotopic Lung Cancer Transplant Model. Therapeutic efficacy was evaluated in an orthotopic lung cancer transplant model using increasing doses of SN38-βGal and SN38-βGlcNAc. The SN38-βGal and SN38-βGlcNAc groups were compared with a vehicle group (DMSO administration) to assess tumor suppression. The vehicle group (n=3), the SN38-βGal group (n=4), and the SN38-βGlcNAc group (n=5) were used in a BALB / cAJcl-nu / nu luciferase-expressing A549 cell peritoneal dissemination mouse model. Administration was intravenous (i.v.). A total of five doses of 40 mg / kg were administered every other day, followed by seven doses of 80 mg / kg every other day. Tumor volume was assessed by evaluating luciferin / luciferase chemiluminescence values. The endpoint was a significant weight loss (20% reduction). Results showed that tumor growth was significantly suppressed in all mice in the SN38-βGal and SN38-βGlcNAc groups compared to the vehicle group. While the vehicle group showed a significant increase in tumor volume after two weeks of treatment, this increase was significantly suppressed in the SN38-βGal and SN38-βGlcNAc groups. Furthermore, the SN38-βGlcNAc group showed a slightly higher tumor-suppressing effect than SN38-βGal, but no significant difference in effect was observed. After evaluation, lungs were removed from the mice to examine tumor volume. Macroscopic findings confirmed a significant reduction in tumor lesions in the treatment group compared to the vehicle group. The fluorescent reagent HMRef-βGlcNAc, which can visualize A549 tumor lesions, was sprayed onto the tumor lesions, and the tumor lesions were visualized and photographed. Significantly fluorescent areas (tumor sites) were confirmed in the vehicle group, while the number of areas showing this fluorescence was significantly reduced in the administration group. Furthermore, when weight transitions were evaluated throughout this study, no significant decrease was observed in either group compared to the vehicle group. Furthermore, no noticeable side effects were observed macroscopically, such as immediately after administration of SN38-βGal or SN38-βGlcNAc.On the other hand, in the CPT-11-treated group (n=3, 40 mg / kg, q2dx12), weight loss was observed during the first two weeks, but weight gain was observed after two weeks. SN38-βGal and SN38-βGlcNAc, administered at the same doses, did not show weight loss during the first two weeks. These results confirmed the therapeutic efficacy of SN38-βGal and SN38-βGlcNAc in a mouse lung cancer model.

[0226] The specific procedure for the therapeutic experiment is as follows: (1) Model mice were prepared using BALB / cAJcl-nu / nu (7 weeks old) as follows: The skin of the chest was incised, the subcutaneous tissue was removed, and 2 × 10 luciferase-expressing A549 cells were injected into the left lung through the intercostal pleura. ７ Cells / mL (50 μL, 5% Matrigel) were injected. After injection, the skin was sutured. (2) One week later, tumor formation was confirmed by luciferin / luciferase chemiluminescence, and intravenous drug administration was initiated. Drugs were prepared by dissolving 200 mM DMSO stock in PBS, and administered in a total volume of 100 μL. Luciferin was administered intraperitoneally at 3 mg (200 μL). After anesthesia with isoflurane, chemiluminescence levels were measured 20 minutes after administration using an in vivo imaging device. (3) On the evaluation day, tumor burden was assessed by luciferin / luciferase chemiluminescence. (4) After treatment, HMRef-βGlcNAc was administered intravenously at 5 mg / kg (100 μL PBS). Thirty minutes after administration, the sternum was opened and the lungs were removed. (5) Fluorescence imaging of cancer lesions was performed using an in vivo fluorescence imaging device, and the results were compared between the vehicle and administration groups.

[0227] Figure 25 shows the results of therapeutic evaluation of SN38-βGal and SN38-βGlcNAc in an A549 lung cancer orthotopic transplant model. Panel a of Figure 25 shows the dosing schedule, and panel b shows the tumor suppression effect in each administration group. Panel c of Figure 25 shows an example of luciferin / luciferase chemiluminescence imaging in each group. Panel d of Figure 25 shows the change in body weight during the treatment period in each group, and panel e of Figure 25 shows the results of fluorescent imaging of lung cancer sites after treatment in each group.

[0228] Example 14 Analysis of Pharmacokinetic Parameters in Common Marmosets Common marmosets (intravenous) SN38-βGal or SN38-βGlcNAc (10 mg / kg) were administered intravenously to common marmosets via 11-minute infusion, and pharmacokinetic parameters were calculated (Figure 26, Tables 9-10). The blood half-life of SN38-βGal was 58.3 minutes, and that of released SN38 was 85.1 minutes. The blood half-life of SN38-βGlcNAc was 47.6 minutes, and that of released SN38 was 93.4 minutes. No significant abnormalities or vomiting were observed in any of the animals after drug administration.

[0229] The specific test procedure is as follows. (1) Three marmosets (male, six animals total) were placed in each of the SN38-βGal and SN38-βGlcNAc groups, and the tails at the injection site were shaved. (2) The base of the tail was avascularized from the injection site to dilate the blood vessels. The veins on both sides of the tail were identified, and the puncture site was disinfected with an alcohol swab. Then, injection was performed using a 27-gauge syringe needle from either the left or right vein. The prepared SN38-βGal or SN38-βGlcNAc solution (1.0 mg / mL) was intravenously administered at a dose of 10 mg / 10 mL / kg using an infusion pump at an infusion rate of 350 μL / min (approximately 11 minutes; the actual injection time τ for each animal was accurately recorded due to differences in body weight). (3) Immediately after the start of infusion, approximately 220 μL of blood was drawn using a heparinized syringe at 5, 15, 30, and 45 minutes, and at 1, 2, 3, and 6 hours. Plasma was collected by centrifugation at 1200 G for 15 minutes at 4°C. (4) After dispensing the plasma into microtubes, 100 μL of the blood was accurately taken and added to microtubes containing 100 μL of 200 μM inhibitor N-(n-nonyl)deoxygalactonoijirimycin solution (SN38-βGal group) or 200 μM inhibitor PUGNAc solution (SN38-βGlcNAc group) and quickly mixed. The resulting plasma was stored frozen at or below -80°C. (5) 50 μL of plasma was mixed with 200 μL of a 1:1 MeOH:MeCN solution (internal standard camptothecin) and allowed to stand for approximately 10 minutes. (6) The supernatant was collected after 15 minutes of centrifugation at 1,700 G and 4°C, and then loaded onto a Cosmos Spin Filter and centrifuged at 12,000 G and 4°C for 5 minutes to recover the deproteinized sample. (7) 50 μL of MiliQ was added to 50 μL of the collected sample, and the MS spectrum was measured by UPLC-MS. (8) The blood concentration was calculated from a calibration curve prepared using a plasma sample in the same manner. The initial concentration was set to 0, and three points, 2, 3, and 6 hours, were selected as the elimination phase to calculate the blood half-life.

[0230] 26 shows the changes in blood concentration after administration of SN38-βGal or SN38-βGlcNAc. 10 mg / kg of the drug was administered intravenously via infusion for 11 minutes. Error bars are SD.

[0231]

[0232]

[0233] Example 15 (1) Cytotoxicity Test of Developed Prodrugs by MTT Assay (H226 Cells) A cytotoxicity test of SN38-βGal and SN38-βGlcNAc was performed on lung squamous cell carcinoma cells, H226, using the MTT assay. As a result, cell proliferation was inhibited with increasing concentrations of SN38-βGal and SN38-βGlcNAc (Figure 27). Under the following measurement conditions, the calculated IC50 values ​​were 1.08 μM for SN38, 1.39 μM for SN38-βGal, 1.50 μM for SN38-βGlcNAc, and 102 μM for CPT-11. SN38-βGal and SN38-βGlcNAc showed lower IC50 values ​​than CPT-11. Furthermore, it was confirmed that the cytotoxicity of SN38-βGal was significantly reduced by the addition of 40 μM N-(n-nonyl)deoxygalactonoijirimycin, an inhibitor of the target enzyme β-galactosidase. The cytotoxicity of SN38-βGlcNAc was significantly reduced by the addition of 40 μM PUGNAc, an inhibitor of the target enzyme β-hexosaminidase. In this study, Cell Proliferation Kit I (MTT), SIGMA-ALDRICH, was used.

[0234] The specific procedure for the cytotoxicity test is as follows: (1) 6.0 × 10 H226 cells were placed in a 96-well plate. ３ 100 μL of RPMI-1640 containing cells, 10% FBS, and 1% PS was added, and the cells were incubated at 37°C and 5% CO ２ (2) 10 μL of medium containing each drug concentration was added, and the cells were incubated at 37°C, 5% CO ２(3) 10 μL of MTT labeling reagent was added, and the mixture was incubated at 37°C and 5% CO for 4 hours. ２ (4) 100 μL of solubilization buffer was added, and the mixture was incubated in a 5% CO ２ The cells were incubated in the same environment for 24 hours. (5) The absorbance at 560 nm was measured to evaluate cell viability, and the IC50 value was calculated.

[0235] Figure 27 shows the results of a cytotoxicity test using an MTT assay. Panel a of Figure 27 shows the cell growth inhibition of lung squamous cell carcinoma H226 cells at each drug concentration, and panel b shows the cell growth inhibition curve and IC50 value for each drug. Panel c of Figure 27 shows the suppression of drug efficacy in the presence of an inhibitor.

[0236] (2) Tumor Growth Inhibition Test in an H226 Subcutaneous Tumor Model SN38-βGlcNAc was administered intravenously at 200 mg / kg three times a week for two weeks to a mouse model bearing subcutaneous tumors of the lung squamous cell carcinoma H226 cells, and the tumor growth inhibitory effect was evaluated. There were five animals in each of the vehicle (DMSO-administered) and SN38-βGlcNAc-administered groups. While significant tumor growth was observed in the vehicle group, tumor growth was almost completely suppressed in the SN38-βGlcNAc-administered group ( Figure 28 ).

[0237] The specific procedure for the therapeutic experiment is as follows: (1) BALB / cAJcl-nu / nu (6-week-old, female) was inoculated with 1.5 × 10 H226 cells. ６ (2) After 31 days, the tumor volume was approximately 50-150 mm ３ Drug administration was initiated for individuals whose tumor size reached approximately 1000 mg / kg. The drug SN38-βGlcNAc was prepared by dissolving a 200 mM DMSO stock in PBS, and administered intravenously via the tail vein in a total volume of 100 μL at a dose of 200 mg / kg three times a week for two weeks. (3) On the day of evaluation, body weight was measured, and tumor volume was evaluated using a vernier caliper. (4) On the 19th day after the start of administration, tumors were excised and their sizes were compared.

[0238] Figure 28 shows the tumor growth inhibitory effect of SN38-βGlcNAc in H226 subcutaneous tumor model mice. Panel a in Figure 28 shows the administration schedule, panel b shows the time course of tumor volume. Panel c in Figure 28 shows the time course of body weight change, and panel d shows a photograph of the excised tumor on Day 19. Error bars = SD.

[0239] Example 16: Growth inhibition test in organoids Growth inhibition tests were conducted on lung cancer organoids using the developed prodrugs SN38-βGal and SN38-βGlcNAc. The organoids were primary lung squamous cell carcinoma organoids established at the Fukushima Medical University Medical-Industry TR Center. RLUN016-2 (F-PDO_000124) was used, which shows low expression of PD-L1 but high expression of HEXB, and has been shown to exhibit gene mutations, fusion genes, and high expression of HEXB in current lung cancer genetic diagnosis (Figure 29b). Growth inhibition tests were conducted on SN38, SN38-βGal, and SN38-βGlcNAc, and the IC50 values ​​were calculated to be 2.70 nM, 284 nM, and 18.0 nM, respectively. Both showed significant cell growth inhibition in organoids (Figure 29a). In particular, SN38-βGlcNAc showed a lower IC50 value than SN38-βGal, suggesting that high expression of HEXB is involved in the growth-suppressing effect of the drug. Furthermore, immunostaining of the target enzymes GLB1, HEXA, and HEXB was performed on PDX tumors established from the same cancer as these organoids, and their expression was confirmed (Figure 29c).

[0240] The specific procedure for the treatment experiment is as follows: (1) Frozen cell stock was thawed in a 37°C water bath for 2 minutes. The cell suspension was collected and transferred to a 50 mL centrifuge tube containing 10 mL of medium. The cells were centrifuged at 200 × g for 3 minutes, and the supernatant was removed. Five mL of medium was added to resuspend the cells, and the cells were then transferred to a 75 cm ２The cells were seeded into a flask and cultured in a CO2 incubator. (2) The cells were collected in a 15 mL centrifuge tube. Centrifuged at 200 × g for 2 minutes, and all but 3 mL of the supernatant was removed. After adding 2 mL of fresh medium and gently pipetting, 5 mL of the cell suspension was poured into a flask containing 10 mL of medium. After adding 4 mL of fresh medium and gently pipetting, 5 mL of the cell suspension was dispensed into two flasks containing 10 mL of medium. (3) All cells were collected in a 15 mL centrifuge tube and centrifuged at 200 × g for 2 minutes. After centrifugation, the cell pellet volume was measured and confirmed to be 150 μL. The supernatant was removed, leaving only 3 mL of medium. Fresh medium was added to bring the cell suspension volume to 15 mL, and the flask was returned to the flask and cultured in a CO2 incubator. (4) Eight concentrations of SN38, SN38-βGal, and SN38-βGlcNAc were prepared from a 100 mM DMSO stock solution. Each solution was dispensed into a 96-well plate and stored frozen at -80°C until use. (5) The required amount of cell culture medium was collected from the flask, and the cell clumps were fragmented using a CellPet FT (70 μm filter diameter), followed by a 30-fold dilution to prepare a cell suspension. 150 μL of the prepared cell suspension was seeded onto a 96-well spheroid plate using a Multi-drop Combi. The cells were cultured in a CO2 incubator until drug addition. (6) The plates containing SN38, SN38-βGal, and SN38-βGlcNAc at the evaluation concentrations and the cell plate prepared by cell suspension preparation were placed in a dispenser, and 0.15 μL of drug was added to each well for N = 3 and 8 concentrations. The final drug treatment concentrations were 100, 20, 4, 0.8000, 0.1600, 0.0320, 0.0064, and 0.0013 μM, and the final DMSO concentration was 0.1%. The plates were cultured in a CO2 incubator (5% CO2, 37°C) for 6 days before ATP measurement. (7) The plates to be measured were removed from the CO2 incubator and allowed to stand at room temperature for 30 minutes. 10 μL of CellTiter-Glo 3D reagent was added to each well. After vortexing for 5 minutes, the plates were allowed to stand at room temperature for 10 minutes. The chemiluminescence value of each plate was measured, and the IC50 value was calculated from the ratio of the ATP amount in each group, assuming the ATP amount in cells to which only DMSO had been added as 100%.

[0241] Figure 29 shows the results of a growth inhibition test in lung squamous cell carcinoma organoids. Figure 29A shows the growth inhibition results in organoids, and Figure 29B shows the lung cancer gene test item information and GLB1, HEXA, and HEXB expression information of the organoids used. Figure 29C shows the immunostaining of GLB1, HEXA, and HEXB in the PDX tumors of the organoids used. Error bars = SD.

[0242] Example 17: Tumor Growth Inhibition Test in PDX Model Mice For SN38-βGlcNAc, which demonstrated high sensitivity in the organoid test, an in vivo tumor growth inhibition test was conducted using a primary lung squamous cell carcinoma PDX tumor (DLUN013 / F_PDX_000162) derived from the same patient and established at the Fukushima Medical University Medical-Industrial TR Center. SN38-βGlcNAc was administered intravenously at 200 mg / kg to PDX subcutaneous tumor model mice six times over a two-week period, as shown in the administration schedule (Figure 30(a)). The tumor growth inhibitory effect was evaluated. The vehicle (DMSO-administered) group and the SN38-βGlcNAc-administered group each had an n=6 population. While significant tumor growth was observed in the vehicle group, tumor growth was significantly suppressed in the SN38-βGlcNAc-administered group (Figure 30(b), d, and e)). Furthermore, no change in body weight was observed following drug administration (Fig. 30c). After tumor removal from the vehicle group, fluorescence imaging using HMRef-βGlcNAc (50 μM, PBS solution) confirmed significant activation of β-hexosaminidase, the target enzyme of SN38-βGlcNAc. The observed fluorescence was significantly suppressed in the presence of PUGNAc (500 μM), a β-hexosaminidase inhibitor (Fig. 30f).

[0243] The specific procedure for the treatment experiment is as follows: (1) Tubes containing frozen tumor tissue were thawed in a 37°C water bath. The tissue fragments were transferred to a dish containing HBSS (+1% PS). (2) NOG (NOD.Cg-Prkdc<scid>Il2rg<tmlSug> / ShiJic) (7-week-old, female) mice were anesthetized with an intraperitoneal injection of an anesthetic (a triple mixture of Domitor, Betorphal, and Midazolam). The hair on the right dorsal region was shaved with clippers, the shaved area was disinfected with 70% ethanol, and then spread with isodine. (3) An approximately 3 mm incision was made at the transplantation site on the right dorsal region, and the tumor tissue was loaded into the tip of a trocar. The trocar was inserted into the transplantation site and the tumor fragments were extruded (approximately 30 mg of tumor fragments were transplanted per mouse). After transplantation, the incision was sealed with medical grade Araldite. An antisedan (atipamezole) solution was administered intraperitoneally, and the anesthesia was released. (4) After 34 days, the tumor volume was approximately 40-250 mm ３ Drug administration was initiated for individuals that reached this level. SN38-βGlcNAc was prepared by dissolving a 400 mM DMSO stock in PBS, and a total volume of 100 μL was administered intravenously via the tail vein at a dose of 200 mg / kg three times a week for two weeks. (5) On the day of evaluation, tumor volume and body weight were measured. (6) After tumor removal, fluorescent imaging was performed using HMRef-βGlcNAc (50 μM, PBS solution) in the presence and absence of PUGNAc (500 μM), a β-hexosaminidase inhibitor.

[0244] Figure 30 shows the tumor growth inhibitory effect of SN38-βGlcNAc in PDX model mice. Panel a in Figure 30 shows the administration schedule, panel b shows the change in tumor volume over time. Panel c in Figure 30 shows the change in body weight over time, and panel d shows the change in tumor volume on Day 25. Panel e in Figure 30 shows an image of the tumor excised on Day 25, and panel f shows the results of evaluating β-hexosaminidase activity by fluorescent imaging of the tumor. Error bars = SD.

[0245] Example 18: Tumor Growth Inhibition Test in a Small Cell Lung Cancer Model The tumor growth inhibitory effects of the developed prodrugs SN38-βGal and SN38-βGlcNAc were evaluated in a small cell lung cancer model. The model was created by orthotopically transplanting SHP77-luc, a small cell lung cancer cell line in which luciferase was expressed, into the lungs of BALB / cAJcl-nu / nu mice. Each of the vehicle (DMSO-administered) group, SN38-βGal-administered group, and SN38-βGlcNAc-administered group consisted of six animals. Administration was intravenous (i.v.) at a dose of 40 mg / kg twice weekly for four weeks. Tumor burden was assessed by the luminescence value of luciferin / luciferase. As a result, significant tumor growth suppression was confirmed in the SN38-βGal and SN38-βGlcNAc groups compared to the vehicle group (Figure 31A). Furthermore, while weight loss in the vehicle group was thought to be due to cancer progression, no weight loss was observed during the treatment period in the SN38-βGal and SN38-βGlcNAc groups (Figure 31B). Furthermore, fluorescent detection of small cell lung cancer using HMRef-βGlcNAc was also confirmed (Figure 32).

[0246] The specific procedure for the therapeutic experiment is as follows. (1) Model mice were created using 20 BALB / cAJcl-nu / nu (7-week-old / female) mice according to the following procedure. The mice were anesthetized by intraperitoneal administration of 150-200 μL of an anesthetic (a triple mixture of Domitor, Betolfar, and Midazolam). The skin on the left chest was incised, the subcutaneous tissue was removed, and 2 × 10 cells of SHP77-luc cell suspension were injected into the left lung through the intercostal pleura. ７Cells / mL (50 μL, 5% Matrigel) were injected. The skin was closed with three sutures, and 150-200 μL of Antisedan (atipamezole) solution was administered intraperitoneally. The mice were then confirmed to be awake. (2) After three weeks, tumor formation was confirmed by luciferin / luciferase luminescence, and drug administration was initiated. The drug was prepared by dissolving a 200 mM DMSO stock in PBS, and a total volume of 100 μL was administered via the tail vein. Luciferin was administered intraperitoneally at 3 mg (200 μL). After anesthesia with isoflurane, luminescence intensity was measured 15 minutes after administration using an in vivo imaging device. (3) On the day of evaluation, body weight and tumor burden were assessed by luciferin / luciferase luminescence.

[0247] Figure 31 shows the results of a treatment experiment in an orthotopic transplant model of small cell lung cancer. Panel a of Figure 31 shows the tumor suppression effect in each administration group, and panel b shows the time course of body weight change. Error bars = SD. Figure 32 shows an example of fluorescent detection of human small cell lung cancer tissue using HMRef-βGlcNAc. Panel a of Figure 32 shows fluorescent images of human normal lung tissue and small cell lung cancer tissue specimens 30 minutes after the addition of HMRef-βGlcNAc (50 μM), and panel b shows immunostaining of HEXA and HEXB in small cell lung cancer tissue.

[0248] Example 19: Examination of the Therapeutic Effect of SN38-βGal in a SKOV3 Ovarian Cancer Peritoneal Dissemination Model (1) Results of the First Treatment Experiment Next, the therapeutic effect of SN38-βGal in a peritoneal dissemination model of ovarian cancer was examined. The vehicle (DMSO) group (n=5) and SN38-βGal at 20 mg / kg (n=4) were administered intraperitoneally at 20 mg / kg, five times a week (with two days off treatment) for four weeks and three days. The results again suggested a tumor growth inhibitory effect compared to the vehicle group. Furthermore, no significant weight loss was observed, suggesting that no significant side effects occurred within this administration range (Figure 33).

[0249] The specific procedure for the therapeutic experiment is as follows: (1) 1.5 × 10 luciferase-expressing SKOV3 cells were injected into the abdominal cavity of BALB / cAJcl-nu / nu (7 weeks old). ６(2) One week later, tumor formation was confirmed by luciferin / luciferase chemiluminescence. (3) For mice in which tumor formation was confirmed, intraperitoneal administration of the drug was initiated in a cycle of five consecutive days of administration per week followed by two days of rest. The drug was prepared by dissolving a 100 mM DMSO stock in PBS, and administered in a total volume of 100 μL. (4) On the day of evaluation, 3 mg of luciferin potassium dissolved in 200 μL of PBS was intraperitoneally administered. After anesthesia with isoflurane, the mice were anesthetized and the luminescence intensity 15 minutes after administration was measured using an in vivo imaging device. Body weight was also measured at the same time.

[0250] Figure 33 shows the results of a first treatment experiment using SN38-βGal in an SKOV3 ovarian cancer peritoneal dissemination model. Panel a in Figure 33 shows the dosing schedule, and panel b shows the evaluation of tumor suppression effect. Panel c in Figure 33 shows the change in body weight over the treatment period. Panel d in Figure 33 shows a comparison of chemiluminescence values ​​from SKOV3 tumors in the vehicle group and the SN38-βGal-administered group. It is shown that SN38-βGal significantly suppressed tumor growth compared to the vehicle group.

[0251] (2) Results of the Second Treatment Experiment Using the same protocol as the first treatment experiment, the vehicle (DMSO) group (n=4) and SN38-βGal 20 mg / kg (n=4) were administered intraperitoneally at 20 mg / kg, five times a week (with two days off) for four weeks and three days. As a result, tumor growth inhibitory effects were again confirmed compared to the vehicle group. Furthermore, no significant weight loss was observed, suggesting that no significant side effects occurred within this dose range (Figure 34).

[0252] Figure 34 shows the results of a second treatment experiment using SN38-βGal in an SKOV3 ovarian cancer peritoneal dissemination model. Panel a in Figure 34 shows the dosing schedule, and panel b shows the evaluation of tumor suppression effect. Panel c in Figure 34 shows the change in body weight over the treatment period. Panel d in Figure 34 shows a comparison of chemiluminescence values ​​from SKOV3 tumors in the vehicle group and the SN38-βGal-administered group. It is shown that SN38-βGal significantly suppressed tumor growth compared to the vehicle group.

[0253] Example 20: Combination Experiment with Anti-PD-1 Antibody The combined effect of SN38-βGlcNAc and an anti-PD-1 antibody, which is currently used as a primary cancer immunotherapy in lung cancer treatment, was investigated. Evaluation was performed on a mouse subcutaneous tumor model of the mouse Lewis lung carcinoma-derived cell line LLC (3LL), which has been reported to be sensitive to anti-PD-1 antibodies. The vehicle (DMSO) group consisted of n=8, the anti-PD-1 antibody administration group consisted of n=5, the SN38-βGlcNAc administration group consisted of n=6, and the anti-PD-1 antibody and SN38-βGlcNAc combination group consisted of n=5. The antibody used was the anti-mouse PD-1 antibody Clone RMP1-14 (Leinco Technologies, Inc.). The anti-PD-1 antibody was administered intraperitoneally at 100 μg twice a week, and SN38-βGlcNAc was administered by retro-orbital vein at 150 mg / kg three times a week. As a result, compared to the vehicle group, the anti-PD-1 antibody administration group, the SN38-βGlcNAc administration group, and the anti-PD-1 antibody and SN38-βGlcNAc combination group showed tumor growth inhibition, with the greatest tumor growth inhibition observed in the anti-PD-1 antibody and SN38-βGlcNAc combination group. Furthermore, no weight loss was observed in any of the groups. These results suggest that SN38-βGlcNAc can be expected to have a greater therapeutic effect when used in combination with an anti-PD-1 antibody (Figure 35).

[0254] The specific procedure for the therapeutic experiment is as follows: (1) After shaving the hair from the upper right leg to the dorsal side of a C57BL / 6JJcl (7-week-old female) with clippers, 2.0 × 10 LLC cells were injected. ６ (2) After 6 days, the tumor volume was approximately 50-170 mm ３ Drug administration was initiated for individuals whose tumor size reached approximately 100 μL. SN38-βGlcNAc was prepared by dissolving a 400 mM DMSO stock in PBS, and administered at a dose of 150 mg / kg three times a week for two weeks in a total volume of 100 μL. Administration was performed intravenously via the orbital vein after anesthesia with isoflurane inhalation. 100 μg of anti-PD-1 antibody (Clone RMP1-14) was dissolved in 100 μL of PBS and administered intraperitoneally. (3) Body weight was measured on the day of evaluation, and tumor volume was assessed using a vernier caliper. (4) When tumor volume reached 2000-3000 mm３ The point at which this was reached was defined as the endpoint.

[0255] Figure 35 shows the results of an experiment using an anti-PD-1 antibody in combination with SN38-βGlcNAc. In Figure 35, "a" shows the administration schedule, and "b" shows the time course of tumor volume. In Figure 35, "c" shows the time course of weight change, and "d" shows the time course of tumor volume for each individual. Error bars = SD.

[0256] (B) Development of a new prodrug-type anticancer drug targeting protease activity Next, as a new prodrug-type anticancer drug targeting protease activity, we synthesized the prodrug KK-SN38, which targets puromycin-sensitive aminopeptidase activity, and evaluated its functionality as a prodrug-type anticancer drug.

[0257] 1. Synthesis of Prodrug Compounds [Synthesis Example 4] Synthesis of KK-SN38 Compound 5 (KK-SN38) of the present invention was synthesized according to the following scheme 4.

[0258] Scheme 4

[0259] (1) Synthesis of Compound 1 1.00 g (4.22 mmol) of 4-((tert-butyldimethylsilyloxy)methyl)aniline, 2.00 g (4.26 mmol) of Fmoc-Lys(Boc)-OH, 1.63 g (12.6 mmol) of DIEA, and 2.40 g (6.32 mmol) of HATU were dissolved in 30 mL of DMF (ultra-dehydrated) and stirred for 2 hours at room temperature under argon atmosphere. 10 mL of AcOEt was added to the reaction solution, which was then washed with 30 mL (x2) of MilliQ and 30 mL (x2) of saturated saline, followed by filtration with anhydrous MgSO . ４ The resulting compound was dried with 15 mL of DMF (ultra-dehydrated) containing 20% ​​piperidine, and stirred for 1 hour at room temperature under argon atmosphere. The reaction solution was washed with 30 mL (x2) of MilliQ and 30 mL (x2) of saturated saline, and then washed with anhydrous MgSO. ４ The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (CH ２ Cl ２The solvent was distilled off under reduced pressure to obtain Compound 1 (1.30 g, 2.79 mmol) as a yellow oil in a combined yield of 66.1% for the two steps. １ The product was identified by HNMR and ESI-HRMS.

[0260] 1 H-NMR (400 MHz, METHANOL-D4) δ7.52 (d, J = 8.2 Hz, 2H), 7.25 (d, J = 8.7 Hz, 2H), 4.68 (s, 2H), 3.39 (t, J=6.4 Hz, 2H), 3.02 (t, J=6.2 Hz, 2H), 1.48-1.38 (m, 15H), 0.91 (s, 9H), 0.07 (s, 6H). HRMS(ESI+): calcd for [M+H]+466.31011; found 466.31273 (2.62 mDa).

[0261] (2) Synthesis of Compound 2: 1.30 g (2.79 mmol) of Compound 1, 1.00 g (2.89 mmol) of Boc-Lys(Boc)-OH, 1.10 g (8.52 mmol) of DIEA, and 1.70 mg (4.47 mmol) of HATU were dissolved in 20 mL of DMF (ultra-dehydrated) and stirred for 2 hours at room temperature under argon atmosphere. 20 mL of AcOEt was added to the reaction solution, which was then washed with 20 mL (x2) of MilliQ and 20 mL (x2) of saturated saline solution. The mixture was then washed with anhydrous MgSO 4 . ４ The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (hexane:AcOEt=60:40→30:70). The solvent was evaporated under reduced pressure to obtain colorless solid Compound 2 (2.08 g, 2.62 mmol) in a yield of 93.7%. １ The product was identified by HNMR and ESI-HRMS.

[0262] 1H-NMR (400 MHz, METHANOL-D4) δ7.51 (d, J = 8.7 Hz, 2H), 7.24 (d, J = 8.7 Hz, 2H), 6.56 (s, 1H), 4.68 (s, 2H), 4.42 (dd, J = 8.5, 5.3 Hz, 1H), 4.00 (dd. 794.50993 (1.02 mDa).

[0263] (3) Synthesis of Compound 3 2.10 g (2.64 mmol) of Compound 2 and 1.40 g (5.36 mmol) of TBAF were dissolved in 30 mL of THF (ultra-dehydrated) and stirred for 1 hour at room temperature under argon atmosphere. 30 mL of AcOEt was added to the reaction solution, which was then washed with 20 mL of MilliQ (×2). After that, anhydrous MgSO was added. ４ The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (hexane:AcOEt=20:80→0:100). The solvent was evaporated under reduced pressure to obtain colorless solid compound 3 (1.20 g, 1.76 mmol) in a yield of 66.7%. １ The product was identified by HNMR and ESI-HRMS.

[0264] 1 H-NMR (400 MHz, METHANOL-D4) δ7.53 (d, J = 8.7 Hz, 2H), 7.27 (d, J = 8.7 Hz, 2H), 4.53 (s, 2H), 4.42 (dd, J = 8.7, 5.5 Hz, 1H), 3.99 (dd, J = HRMS(ESI+): calcd for [M+Na]+702.40547; found 702.40540 (0.08 mDa).

[0265] (4) Synthesis of Compound 4 Compound 3, 1.20 g (1.76 mmol), PBr ３ 240 mg (0.886 mmol) in 20 mL of CH ２ Cl ２ (ultra-dehydrated), stirred at room temperature under argon for 1 hour, and then added NaHCO ３ The reaction mixture was quenched with 10 mL of aqueous solution. The reaction mixture was washed with 30 mL (x2) of MilliQ and 30 mL of saturated saline, and then with anhydrous MgSO ４ The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (hexane:AcOEt=50:50→20:80) to obtain Compound 4 in a yield of 46%. １ The product was identified by HNMR and ESI-HRMS.

[0266] 1 H-NMR (400 MHz, METHANOL-D4) δ7.57 (d, J = 8.2 Hz, 2H), 7.36 (d, J = 8.7 Hz, 2H), 4.55 (s, 2H), 4.44 (dd, J = 8.5, 5.3 Hz, 1H), 4.10 (q, J = HRMS(ESI+): calcd for [M+Na]+766.31926; found 766.31895 (0.31 mDa).

[0267] (5) Synthesis of Compound 5 (KK-SN38) Compound 4, 150 mg (0.202 mmol), SN38, 55 mg (0.140 mmol), Cs ２ CO ３ 70 mg (0.215 mmol) of the product was dissolved in 5 mL of DMF (ultra-dehydrated) and stirred for 1 hour at room temperature under argon atmosphere. 5 mL of AcOE was added to the reaction solution, which was then washed with 5 mL (x2) of MilliQ and 5 mL of saturated saline, followed by anhydrous MgSO ４ The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (CH ２ Cl ２The solvent was evaporated under reduced pressure to give a crude SN38 adduct. This was then purified with 5 mL of CH4Cl4 containing 25% TFA. ２ Cl ２ The mixture was dissolved in ultra-dehydrated bromine and stirred for 3 hours under ice-cooling and argon purging. The solvent was distilled off under reduced pressure, purified by HPLC, and then freeze-dried to obtain the target compound 5, KK-SN38 (83 mg, 0.110 mmol) as a yellow solid in a combined yield of 54.5% for the two steps. １ H NMR, １３ The product was identified by C-NMR and ESI-HRMS.

[0268] 1 H-NMR (400 MHz, DMSO-D6) δ8.00 (d, J = 8.7 Hz, 1H), 7.63 (s, 2H), 7.48 (m, 4H), 7.25 (s, 1H), 5.38 (dd, J = 25.2, 16.0 Hz, 2H), 5.24 (s, 2H), 5.16 (s, 2H), 4.40 (dd, J = 13.3, 7.8 Hz, 1H), 3.85 (m, 1H), 3.08 (m, 2H), 2.75 (m, 4H), 1.84-1.37 (m, 14H), 1.22 (t, J = 7.5 Hz, 3H), 0.85 (t, J = 7.3 Hz, 3H). 13 C-NMR (101 MHz, DMSO-D6) δ173.1, 170.7, 169.1, 157.7, 157.4, 150.6, 150.2, 146.8, 145.0, 144.4, 139.2, 132.0, 129.4, 129.0, 128.3, 123.2, 119.7, 118.8, 116.0, 104.2, 96.6, 72.9, 70.1, 65.8, 54.1, 52.3, 50.1, 46.1, 39.0, 32.0, 31.0, 30.7, 27.3, 27.0, 23.0, 22.8, 21.5, 14.0, 8.3. HRMS(ESI+): calcd for [M+H]+754.39667; found 754.39282 (3.85 mDa).

[0269] Example 21: KK-SN38 Therapeutic Experiment in a Peritoneal Dissemination Model (1) Results of the First Therapeutic Experiment The therapeutic effect of KK-SN38, a prodrug-type anticancer drug having KK, the PSA target sequence, as its substrate, in an ovarian cancer SKOV3 peritoneal dissemination model was examined. The groups were divided into a vehicle (DMSO) group (n = 5) and a KK-SN38 group (n = 4). KK-SN38 was administered intraperitoneally at 10 mg / kg, five times a week (with two days off) for the first week, and then at 5 mg / kg, five times a week (with two days off) for three weeks and three days. The results suggested a tumor growth inhibitory effect compared to the vehicle group. Furthermore, no significant weight loss was observed, suggesting the absence of significant side effects (Figure 36).

[0270] The specific procedure for the therapeutic experiment is as follows: (1) 1.5 × 10 luciferase-expressing SKOV3 cells were injected into the abdominal cavity of BALB / cAJcl-nu / nu (8 weeks old). ６ (2) One week later, tumor formation was confirmed by luciferin / luciferase chemiluminescence. (3) For mice in which tumor formation was confirmed, intraperitoneal administration of the drug was initiated in a cycle of five consecutive days of administration per week followed by two days of rest. The drug was prepared by dissolving a 100 mM DMSO stock in PBS, and administered intraperitoneally in a total volume of 100 μL. (4) On the day of evaluation, 3 mg of luciferin potassium dissolved in 200 μL of PBS was administered intraperitoneally. After anesthesia with isoflurane, the luminescence intensity 15 minutes after administration was measured using an in vivo imaging device. Body weight was also measured at the same time.

[0271] Figure 36 shows the results of a first treatment experiment using KK-SN38 in an SKOV3 ovarian cancer peritoneal dissemination model. Panel a in Figure 36 shows the dosing schedule, and panel b shows the evaluation of tumor suppression effect. Panel c in Figure 36 shows the change in body weight over the treatment period. Panel d in Figure 36 shows a comparison of chemiluminescence values ​​from SKOV3 tumors in the vehicle group and the KK-SN38-administered group. It is shown that KK-SN38 significantly suppressed tumor growth compared to the vehicle group.

[0272] (2) Results of the Second Treatment Experiment The therapeutic effect of KK-SN38 in an ovarian cancer peritoneal dissemination model was examined. The vehicle (DMSO) group (n=4) and KK-SN38 (n=7) were administered intraperitoneally at 5 mg / kg KK-SN38 five times a week (with two days off) for four weeks and three days. The results again suggested a tumor growth inhibitory effect compared to the vehicle group. Furthermore, no significant weight loss was observed, suggesting that no significant side effects occurred within this administration range (Figure 37).

[0273] The specific procedure for the therapeutic experiment is as follows: (1) 1.5 × 10 luciferase-expressing SKOV3 cells were injected into the abdominal cavity of BALB / cAJcl-nu / nu (7 weeks old). ６ (2) One week later, tumor formation was confirmed by luciferin / luciferase chemiluminescence. (3) For mice in which tumor formation was confirmed, intraperitoneal administration of the drug was initiated in a cycle of five consecutive days of administration per week followed by two days of rest. The drug was prepared by dissolving a 100 mM DMSO stock in PBS, and administered in a total volume of 100 μL. (4) On the day of evaluation, 3 mg of luciferin potassium dissolved in 200 μL of PBS was intraperitoneally administered. After anesthesia with isoflurane, the mice were anesthetized and the luminescence intensity 15 minutes after administration was measured using an in vivo imaging device. Body weight was also measured at the same time.

[0274] Figure 37 shows the results of a second treatment experiment using KK-SN38 in an SKOV3 ovarian cancer peritoneal dissemination model. Panel a in Figure 37 shows the dosing schedule, and panel b shows the evaluation of tumor suppression effect. Panel c in Figure 37 shows the change in body weight over the treatment period. Panel d in Figure 37 shows a comparison of chemiluminescence values ​​from SKOV3 tumors in the vehicle group and the KK-SN38-administered group. It is shown that KK-SN38 significantly suppressed tumor growth compared to the vehicle group.

[0275] (C) Prodrug-type anticancer drugs containing various anticancer drugs Next, novel prodrug-type anticancer drugs of the present invention were synthesized by introducing a substrate moiety that targets cancer-specific enzyme activity into the molecule of an anticancer drug other than SN38, and their functionality as prodrug-type anticancer drugs was evaluated.

[0276] 1. Synthesis of Prodrug Compounds (1) Synthesis of Compound 6 Compound 6 was synthesized according to the following scheme.

[0277] p-Hydroxybenzaldehyde (3.00 g, 24.6 mmol), 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-D-glucopyranosyl chloride (9.00 g, 24.6 mmol), Ag ２ O (17.0 g, 73.4 mmol) was dissolved in 80.0 mL of acetonitrile and stirred at room temperature for 20 hours. After filtration through Celite, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (CH ２ Cl ２ The resulting product was purified with a gradient of 100:0 to 70:30 (MeOH:MeOH) to give Compound 6 (10.53 g, 23.3 mmol) as a white solid in a yield of 95.0%. １ H NMR, １３ The product was identified by CNMR and ESI-HRMS.

[0278] 1 H NMR (400 MHz, MeOD): δ 9.90 (s, 1H), 7.91 (d, J = 8.8 Hz, 2H), 7.21 (d, J = 8.8 Hz, 2H), 5.49 (d, J = 8.4 Hz, 1H), 5.39 (t, J = 9.2 Hz, 1H), 5.11 (t, J = 10 Hz, 1H), 4.34 (dd, J = 5.2 Hz, 12.4 Hz, 1H), 4.20-4.10 (m, 3H), 2.07 (s, 3H), 2.06 (s, 3H), 2.04 (s, 3H), 1.93 (s, 3H). 13 C NMR (101 MHz, MeOD): δ192.8, 173.7, 172.3, 171.8, 171.3, 163.2, 133.1, 132.9, 117.8, 98.9, 73.7, 73.3, 69.9, 63.1, 55.4, 22.7, 20.6, 20.6, 20.5. ESI-HRMS (ESI +) m / z calcd. for [M+Na] +, 474.13707; found, 474.13640.

[0279] (2) Synthesis of Compound 7 Compound 7 was synthesized according to the following scheme.

[0280] Compound 6 (750 mg, 1.66 mmol), NaBH ４ (50.0 mg, 1.32 mmol) was dissolved in a mixed solvent of 5.0 mL of dichloromethane and 5.0 mL of methanol, and stirred at room temperature for 1.5 hours. Purified water was added, and the mixture was washed with dichloromethane to extract the target product. The organic phase was washed with saturated brine, dried over sodium sulfate, and filtered, and the solvent was evaporated under reduced pressure to obtain compound 7 (665 mg, 1.46 mmol) as a white solid in a yield of 88.3%. The target product obtained was １ Identification was carried out by HNMR.

[0281] 1 H NMR (400 MHz, CD2Cl2): δ7.33 (d, J = 8.4 Hz, 2H), 7.02 (d, J = 8.4 Hz, 2H), 5.70 (d, J = 8.4 Hz, 1H), 5.43 (t, J = 10 Hz, 1H), 5.31 (d, J = 8.0 Hz, 1H), 5.13 (t, J = 9.6 Hz, 1H), 4.30 (dd, J = 5.6 Hz, 12.0 Hz, 1H), 4.18-4.00 (m, 2H), 3.95-3.85 (m, 1H), 2.09-2.07 (m, 9H), 1.95 (s, 3H). ESI-HRMS (ESI +) m / z Calculate for [M+Na] + , 476.15272; found, 476.15320.

[0282] (3) Synthesis of Compound 8 Compound 8 was synthesized according to the following scheme.

[0283] Compound 7 (278 mg, 0.614 mmol), p-nitrophenyl chloroformate (368 mg, 1.84 mmol), and pyridine (148 μL) were dissolved in 30.0 mL of ethyl acetate and stirred at room temperature under an argon atmosphere for 3 hours. A saturated sodium bicarbonate solution was added, and the mixture was washed with purified water to extract the ethyl acetate phase. The organic phase was washed with saturated brine, dried over sodium sulfate, and filtered, followed by distilling off the solvent under reduced pressure. Purification was performed by silica gel column chromatography (AcOEt:hexane = gradient from 0:100 to 100:0), and compound 8 (267 mg, 0.424 mmol) was obtained as a white solid in a yield of 69.1%. The target product obtained was １ The product was identified by HNMR and ESI-HRMS.

[0284] 1 H NMR (400 MHz, CD2Cl2): δ8.30 (d, J = 9.2 Hz, 2H), 7.44-7.41 (m, 4H), 7.07 (d, J = 8.8 Hz, 2H), 5.78 (d, J = 8.8 Hz, 1H), 5.48 (t, J = 9.6 Hz, 1H), 5.39 (d, J = 8.4 Hz, 1H), 5.27 (s, 2H), 5.14 (t, J = 9.6 Hz, 1H), 4.31 (dd, J = 5.6 Hz, 12.4 Hz, 1H), 4.19-4.07 (m, 2H), 3.97-3.92 (m, 1H), 2.08-2.07 (m, 9H), 1.95 (s, 3H). ESI-HRMS (ESI +) m / z calcd. for [M+Na] + , 641.15892; found, 641.16136.

[0285] (4) Synthesis of Compound 9 Compound 9 was synthesized according to the following scheme.

[0286] Compound 7 (565 mg, 1.25 mmol), PBr ３(337 mg, 1.84 mmol) was dissolved in 20.0 mL of diethyl ether and stirred at room temperature for 1 hour. Saturated sodium bicarbonate solution and ethyl acetate were added, and the mixture was washed with purified water to extract the ethyl acetate phase. The organic phase was washed with saturated saline and then dried over sodium sulfate. After filtration, the solvent was distilled off under reduced pressure to obtain compound 9 (440 mg, 0.854 mmol) as a white solid in a 68.5% yield. The obtained target product was identified by ESI-HRMS. ESI-HRMS (ESI +) m / z calcd. for [M+Na] + , 538.06904; found, 538.06832.

[0287] Synthesis Example 5 Synthesis of DOX-βGlcNAc DOX-βGlcNAc, a compound of the present invention, was synthesized according to the following scheme.

[0288] Compound 8 (170 mg, 0.275 mmol), doxorubicin (150 mg, 0.276 mmol), and triethylamine (600 μL) were dissolved in 8.0 mL of N,N-dimethylformamide and stirred at room temperature for 1 hour. The solvent was evaporated under reduced pressure, diethyl ether was added, and the red precipitate was collected by filtration. The red precipitate was dissolved in 20.0 mL of methanol, NaOMe (149 mg, 2.76 mmol) was added, and the mixture was stirred at room temperature for 1 hour. Amberlite IR120 (300 mg) was added, and after stirring for 10 minutes, the mixture was removed by suction filtration, and the solvent was evaporated under reduced pressure. Silica gel column chromatography (CH ２ Cl ２ The resulting product was purified using a gradient of 100:0 to 75:25 (MeOH:COOH) to give DOX-βGlcNAc (69.0 mg, 0.0770 mmol) as a red solid in a yield of 30.0%. １ H NMR, １３ The product was identified by CNMR and ESI-HRMS.

[0289] 1H NMR (400 MHz, MeOD): δ7.83 (d, J = 7.2 Hz, 1H), 7.75 (t, J = 8.0 Hz, 1H), 7.47 (d, J = 8.0 Hz, 1H), 7.22 (d, J = 8.4 Hz, 2H), 6.93 (d, J = 8.4 Hz, 2H), 5.38 (s, 1H), 5.20-4.94 (m, overlaps with HOD), 4.74 (s, 1H), 4.31-4.24 (m, 1H), 3.98 (s, 3H), 3.90-3.81 (m, 4H), 3.74-3.51 (m, 9H), 4.77 (d, J = 4.4 Hz, 3H), 3.08-3.03 (m, 1H), 2.93-2.87 (m, 1H), 2.37-2.33 (m, 1H), 2.15-2.13 (m, 1H), 2.05-1.94 (m, 4H), 1.76-1.73 (m, 1H), 1.26 (d, J = 6.4 Hz, 3H). 13 C NMR (101 MHz, MeOD): δ214.0, 187.8, 187.5, 173.9, 162.3, 158.9, 158.0, 157.3, 156.1, 137.1, 136.2, 135.6, 135.0, 132.3, 130.6, 129.5, 121.3, 120.5, 120.2, 117.6, 112.3, 112.1, 102.2, 100.8, 78.2, 77.4, 75.8, 71.9, 71.8, 70.2, 68.7, 67.1, 65.8, 62.6, 57.4, 57.1, 37.3, 34.0, 30.9, 23.0, 17.3. ESI-HRMS (ESI +) m / z calcd. for [M+Na] + , 919.27435; found, 919.27385.

[0290] Synthesis Example 6 Synthesis of EPI-βGlcNAc EPI-βGlcNAc, a compound of the present invention, was synthesized according to the following scheme. Compound 8 (85 mg, 0.138 mmol), epirubicin (75 mg, 0.138 mmol), and triethylamine (300 μL) were dissolved in 4.0 mL of N,N-dimethylformamide and stirred at room temperature for 1 hour. The solvent was evaporated under reduced pressure, diethyl ether was added, and the red precipitate was collected by filtration. The red precipitate was dissolved in 20.0 mL of methanol, NaOMe (74.6 mg, 2.76 mmol) was added, and the mixture was stirred at room temperature for 1 hour. Amberlite IR120 (300 mg) was added, and after stirring for 10 minutes, the mixture was removed by suction filtration, and the solvent was evaporated under reduced pressure. Silica gel column chromatography (CH ２ Cl ２ The product was purified using a gradient of 100:0 to 75:25 (MeOH:COOH) to give EPI-βGlcNAc (11.5 mg, 0.0128 mmol) as a red solid in a yield of 9.3%. １ The product was identified by HNMR and ESI-HRMS.

[0291] 1 H NMR (400 MHz, MeOD): δ 7.87 (d, J = 7.2 Hz, 1H), 7.77 (t, J = 8.4 Hz, 1H), 7.49 (d, J = 8.4 Hz, 1H), 7.22 (d, J = 7.6 Hz, 2H), 6.94 (d, J = 8.0 Hz, 2H), 5.35 (s, 1H), 5.20-4.89 (m, overlaps with HOD), 3.99 (s, 3H), 3.93-3.85 (m, 4H), 3.74-3.51 (m, 9H), 3.39 (d, J = 5.2 Hz, 3H), 3.11-3.02 (m, 4H), 2.44-2.42 (m, 1H), 2.08-2.06 (m, 1H), 1.98-1.95 (m, 4H), 1.71-1.65 (m, 1H), 1.30 (d, J = 6.0 Hz, 3H). ESI-HRMS (ESI +) m / z calcd. for [M+Na] + , 919.27435; found, 919.27700.

[0292] Synthesis Example 7 Synthesis of Mel-βGlcNAc Mel-βGlcNAc, a compound of the present invention, was synthesized according to the following scheme. Compound 8 (203 mg, 0.328 mmol), melphalan (100 mg, 0.328 mmol), and triethylamine (400 μL) were dissolved in 15.0 mL of N,N-dimethylformamide and stirred at room temperature for 1.5 hours. The solvent was evaporated under reduced pressure, and the mixture was dissolved in 20.0 mL of methanol. NaOMe (177 mg, 3.28 mmol) was added and stirred at room temperature for 1 hour. Amberlite IR120 (300 mg) was added, and after stirring for 10 minutes, the mixture was removed by suction filtration, and the solvent was evaporated under reduced pressure. Purification by HPLC gave Mel-βGlcNAc (92.6 mg, 0.141 mmol) as a white solid in a yield of 43.0%. The target product obtained was １ H NMR, １３ The product was identified by CNMR and ESI-HRMS.

[0293] 1 H NMR (400 MHz, MeOD): δ7.24 (d, J = 8.8 Hz, 2H), 7.08 (d, J = 8.4 Hz, 2H), 7.00 (d, J = 8.4 Hz, 2H), 6.65 (d, J = 8.4 Hz, 2H), 5.06 (d, J = 8.4 Hz, 1H), 4.33-4.30 (m, 1H), 3.98-3.91 (m, 2H), 3.76-3.57 (m, 10H), 3.47 (s, 2H), 3.33 (t, J = 1.2 Hz, 1H), 3.08 (dd, J = 4.8 Hz, J = 14 Hz, 1H), 3.08 (dd, J = 4.8 Hz, J = 14 Hz, 1H), 2.85 (dd, J = 8.4 Hz, J = 14 Hz, 1H), 2.66 (s, 3H), 2.00 (s, 2H). 13C NMR (101 MHz, MeOD): δ176.5, 174.0, 158.8, 158.3, 146.5, 132.3, 131.6, 130.5, 127.3, 118.0, 117.6, 113.3, 100.8, 78.2, 75.8, 71.8, 67.2, 62.5, 57.7, 57.3, 54.5, 41.8, 40.5, 37.9, 23.1. ESI-HRMS (ESI +) m / z calcd. for [M+Na] + , 680.17482; found, 680.17122.

[0294] Synthesis Example 8 Synthesis of EXT-βGlcNAc EXT-βGlcNAc, a compound of the present invention, was synthesized according to the following scheme.

[0295] Compound 8 (170 mg, 0.275 mmol), exatecan (119 mg, 0.274 mmol), 1-hydroxybenzotriazole (HOBt) (37.0 mg, 0.274 mmol), and pyridine (1.0 mL) were dissolved in 4.0 mL of N,N-dimethylformamide, and the solution was stirred at room temperature under an argon atmosphere for 20 hours. The solvent was evaporated under reduced pressure, and the resulting solution was purified by silica gel column chromatography (CH ２ Cl ２ The resulting product was purified using a gradient of 100:0 to 65:35 NaOH / MeOH to obtain an acetylated glycoside of Exatecan. This was dissolved in 30.0 mL of methanol, and NaOMe (148 mg, 2.74 mmol) was added and stirred at room temperature for 1 hour. Amberlite IR120 (300 mg) was added, and after stirring for 10 minutes, the product was removed by suction filtration, and the solvent was distilled off under reduced pressure. The product was purified by HPLC to obtain EXT-βGlcNAc (32.0 mg, 0.0406 mmol) as a brown solid in a yield of 14.8%. The target product was １ H NMR, １３ The product was identified by CNMR and ESI-HRMS.

[0296] 1H NMR (400 MHz, MeOD): δ7.40-7.33 (m, 3H), 7.23 (d, J = 10.4 Hz, 1H), 7.00 (d, J = 8.0 Hz, 2H), 5.45 (d, J = 16 Hz, 1H), 5.30-5.13 (m, 3H), 5.05 (d, J = 8.4 Hz, 1H, overlaps with HOD), 4.69 (d, J = 18.8 Hz, 1H), 3.95-3.90 (m, 2H), 3.75 (dd, J = 4.4 Hz, J = 11.6 Hz, 1H), 3.62-3.58 (m, 1H), 3.46-3.44 (m, 2H), 3.12-3.08 (m, 1H), 2.99-2.94 (m, 1H), 2.22 (s, 3H), 2.15 (d, J = 16 Hz, 2H), 2.00 (s, 3H), 1.93 (d, J = 6.8 Hz, 2H), 1.00 (t, J = 6.8Hz, 3H). 13 C NMR (101 MHz, MeOD): δ174.6, 173.9, 162.2, 159.0, 158.5, 158.3, 152.8, 152.3, 149.0, 148.8, 146.2, 142.4, 137.3, 132.1, 130.8, 127.0, 125.9, 125.8, 122.6, 120.8, 117.8, 116.5, 110.5, 100.9, 99.2, 78.3, 75.8, 74.1, 71.9, 67.9, 66.8, 62.6, 57.4, 50.9, 32.3, 29.7, 25.4, 23.1, 11.2, 8.3. ESI-HRMS (ESI +) m / z calcd. for [M+Na] + , 811.25972; found, 811.26103.

[0297] Synthesis Example 9 Synthesis of MMAE-βGlcNAc MMAE-βGlcNAc, a compound of the present invention, was synthesized according to the following scheme.

[0298] Compound 8 (70 mg, 0.113 mmol), monomethyl auristatin E (MMAE) (79.7 mg, 0.113 mmol), 1-hydroxybenzotriazole (HOBt) (15.3 mg, 0.113 mmol), N,N-diisopropylethylamine (DIEA) (30 μL), and pyridine (1.5 mL) were dissolved in 6.0 mL of N,N-dimethylformamide and stirred at room temperature under an argon atmosphere for 20 hours. The solvent was removed under reduced pressure, and the mixture was purified by HPLC to obtain an acetylated glycoadduct. This was dissolved in 10.0 mL of methanol, and NaOMe (61.0 mg, 1.13 mmol) was added. The mixture was stirred at room temperature for 1 hour. Amberlite IR120 (300 mg) was added, stirred for 10 minutes, and then removed by suction filtration. The solvent was then removed under reduced pressure. Purification by HPLC gave a colorless solid, MMAE-βGlcNAc (115 mg, 0.107 mmol), in a yield of 94.9%. １ The product was identified by HNMR and ESI-HRMS.

[0299] 1H NMR (400 MHz, MeOD): δ7.43-7.30 (m, 6H), 7.25-7.21 (m, 1H), 7.04 (d, J = 8.0 Hz, 2H), 5.60-5.40 (m, overlaps with HOD), 5.19-5.07 (m, overlaps with HOD), 4.72-4.66 (m, 1H), 4.65-4.58 (m, 1H), 4.28-4.21 (m, 3H), 4.150-4.09 (m, 1H), 3.98-3.89 (m, 3H), 3.80-3.74 (m, 2H), 3.67-3.62 (m, 1H), 3.60-3.50 (m, 1H), 3.48 (d, J = 5.2 Hz, 3H), 3.43-3.29 (m, 10H), 3.25-3.15 (m, 1H), 3.12 (s, 1H), 2.99-2.92 (m, 3H), 2.53-2.48 (m, 2H), 2.30-2.10 (m, 3H), 2.00 (d, J = 4.0 Hz, 4H), 1.91-1.84 (m, 2H), 1.75-1.71 (m, 1H), 1.64-1.59 (m, 1H), 1.50-1.35 (m, 2H), 1.21-1.14 (m, 6H), 1.03-0.75 (m, 19H). ESI-HRMS (ESI +) m / z calcd. for [M+Na] + , 1093.60434; found, 1093.60145.

[0300] Synthesis Example 10 Synthesis of MMAF-βGlcNAc MMAF-βGlcNAc, a compound of the present invention, was synthesized according to the following scheme.

[0301] Compound 8 (127 mg, 0.205 mmol), monomethyl auristatin F (MMAF) (150 mg, 0.205 mmol), 1-hydroxybenzotriazole (HOBt) (27.8 mg, 0.206 mmol), N,N-diisopropylethylamine (DIEA) (53 μL), and pyridine (1.5 mL) were dissolved in 6.0 mL of N,N-dimethylformamide and stirred at room temperature under an argon atmosphere for 20 hours. The solvent was removed under reduced pressure, and the resulting product was purified by HPLC to obtain an acetylated glycoadduct. This product was dissolved in 10.0 mL of methanol, and NaOMe (110 mg, 2.04 mmol) was added. The mixture was stirred at room temperature for 1 hour. Amberlite IR120 (300 mg) was added, stirred for 10 minutes, and then removed by suction filtration. The solvent was then removed under reduced pressure. The product was purified by HPLC to obtain a colorless solid, MMAF-βGlcNAc (34.0 mg, 0.0313 mmol), in a yield of 15.3%. １ H NMR, １３ The product was identified by CNMR and ESI-HRMS.

[0302] 1H NMR (400 MHz, MeOD): δ7.40-7.26 (m, 6H), 7.24-7.21 (m, 1H), 7.05-7.02 (m, 2H), 5.35-5.28 (m, overlaps with HOD), 5.20-5.06 (m, overlaps with HOD), 4.77-4.69 (m, 2H), 4.26-4.21 (m, 1H), 4.15-4.00 (m, 3H), 4.00-3.91 (m, 3H), 3.76-3.72 (m, 3H), 3.63-3.58 (m, 2H), 3.47-3.45 (m, 4H), 3.40-3.30 (m, 10H), 3.30 (s, 2H), 3.11 (s, 1H), 2.97-2.90 (m, 3H), 2.60-2.56 (m, 1H), 2.51-2.49 (m, 1H), 2.38-2.33 (m, 2H), 2.22-2.20 (m, 2H), 2.00 (s, 4H), 1.91-1.88 (m, 1H), 1.80-1.75 (m, 1H), 1.45-1.35 (m, 2H), 1.08-0.88 (m, 25H). 13 C NMR (101 MHz, MeOD): δ176.4, 176.2, 174.7, 174.6, 174.0, 172.1, 159.1, 138.7, 138.6, 132.2, 131.1, 130.6, 130.3, 130.3, 129.4, 129.4, 127.8, 117.8, 100.8, 87.5, 83.5, 78.2, 75.8, 71.9, 68.3, 65.9, 62.6, 62.2, 61.5, 60.9, 58.5, 58.4, 57.4, 56.1, 54.4, 54.3, 45.5, 45.4, 38.4, 33.0, 31.9, 30.3, 27.8, 27.0, 26.9, 25.9, 25.8, 24.5, 23.0. ESI-HRMS (ESI +) m / z calcd. for [M+Na] + , 1107.58221; found, 1107.58360.

[0303] Synthesis Example 11 Synthesis of NGT-βGlcNAc NGT-βGlcNAc, a compound of the present invention, was synthesized according to the following scheme.

[0304] Compound 9 (168 mg, 0.326 mmol), nogitecan (164 mg, 0.340 mmol), Cs ２ CO ３ (1.0 g, 3.07 mmol) was dissolved in 30.0 mL of acetonitrile and stirred at room temperature for 3 hours. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (CH ２ Cl ２ The acetylated sugar adduct of Nogitecan was obtained by purifying the product using a gradient of 100:0 to 70:30 NaOH / MeOH. This was dissolved in 10.0 mL of methanol, and NaOMe (88.1 mg, 1.63 mmol) was added and stirred at room temperature for 1 hour. Amberlite IR120 (300 mg) was added, and after stirring for 10 minutes, the product was removed by suction filtration, and the solvent was distilled off under reduced pressure. The product was purified by HPLC to obtain a yellow solid, NGT-βGlcNAc (3.6 mg, 0.00493 mmol), in a yield of 1.5%. The target product was １ The product was identified by HNMR and ESI-HRMS.

[0305] 1H NMR (400 MHz, MeOD): δ8.88 (s, 1H), 8.37 (d, J = 9.6 Hz, 1H), 7.98 (d, J = 9.6 Hz, 1H), 7.50 (s, 1H), 7.52 (d, J = 8.4 Hz, 2H), 7.09 (d, J = 8.8 Hz, 2H), 5.58 (d, J = 16.4 Hz, 1H), 5.44-5.36 (m, overlaps with HOD), 5.10 (d, J = 8.8 Hz, overlaps with HOD), 4.90-3.87 (m, 3H), 3.71-3.57 (m, 5H), 3.48-3.92 (m, overlaps with MeOD), 2.95 (s, 6H), 2.05-1.90 (m, 5H), 1.01 (t, J = 7.2 Hz, 3H). ESI-HRMS (ESI +) m / z calcd. for [M+Na] + , 753.27423; found, 743.27355.

[0306] Synthesis of Compound 10 Compound 10 was synthesized according to the following scheme.

[0307] Compound 8 (200 mg, 0.324 mmol), N,N'-dimethylethylenediamine (114 mg, 1.29 mmol), and triethylamine (2.0 mL) were dissolved in 5.0 mL of N,N-dimethylformamide and stirred at room temperature for 2 hours. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (CH ２ Cl ２ The resulting mixture was purified using a gradient of 100:0 to 0:100 (MeOH: 100:0 to 0:100) to give compound 10 (161 mg, 0.284 mmol) as a white solid in a yield of 87.7%. １ H NMR, １３ The product was identified by CNMR and ESI-HRMS.

[0308] 1H NMR (400 MHz, MeOD): δ7. 35(d, J = 8.4 Hz, 2H), 7.04 (d, J = 8.8 Hz, 2H), 5.40-5.33 (m, 2H), 5.11-5.07 (m, 3H), 4.34 (dd, J = 5.2 Hz, J = 12.4 Hz, 1H), 4.18-4.11 (m, 2H), 4.05-4.02 (m, 1H), 3.43 (s, 2H), 2.94 (s, 3H), 2.75-2.70 (m, 2H), 2.41 (s, 2H), 2.35 (s, 2H), 2.06-2.03 (m, 9H), 1.93 (s, 3H). 13 C NMR (101 MHz, MeOD): δ172.1, 170.8, 170.4, 169.8, 157.0, 131.3, 129.5, 129.2, 116.4, 98.4, 72.4, 71.6, 68.7, 66.5, 61.8, 54.1, 34.5, 33.9, 33.4, 21.3, 19.2, 19.2, 19.2. ESI-HRMS (ESI +) m / z calcd. for [M+Na] + , 590.23203; found, 590.23179.

[0309] Synthesis of Compound 11 Compound 11 was synthesized according to the following scheme.

[0310] Etoposide (590 mg, 1.00 mmol) and p-nitrophenyl chloroformate (200 mg, 1.00 mmol) were dissolved in 30.0 mL of tetrahydrofuran and stirred at room temperature for 1 hour. The precipitate was removed by suction filtration, and the solvent was evaporated under reduced pressure. Purification was carried out by silica gel column chromatography (AcOEt:hexane = 0:100 to 80:20 gradient) to obtain colorless solid compound 11 (410 mg, 0.544 mmol) in a yield of 54.3%. The target product obtained was １ H NMR, １３ The product was identified by CNMR and ESI-HRMS.

[0311] 1H NMR (400 MHz, CD2Cl2): δ8.18 (dd, J = 2.0 Hz, J = 6.8 Hz, 2H), 7.08 (dd, J = 2.4 Hz, J = 7.2 Hz, 2H), 6.78 (d, J = 10 Hz, 1H), 6.45 (d, J = 2.5 Hz, 1H), 6.27 (s, 2H), 5.91 (d, J = 8.4 Hz, 2H), 5.24 (s, 1H), 4.84 (d, J = 3.2 Hz, 1H), 4.64 (d, J = 5.2 Hz, 1H), 4.57-4.55 (m, 1H), 4.32-4.30 (m, 1H), 4.14 (t, J = 8.4 Hz, 1H), 4.08-4.05 (m, 1H), 3.66 (s, 6H), 3.54-3.44 (m, 3H), 3.28-3.15 (m, 4H), 3.04 (s, 1H), 2.84 (m, 1H), 1.24 (d, J = 5.2 Hz, 1H). 13 C NMR (101 MHz, CD2Cl2): δ175.1, 156.0, 151.6, 150.7, 149.3, 147.8, 146.9, 145.9, 139.5, 132.4, 129.3, 128.3, 125.7, 123.1, 110.8, 109.6, 108.0, 102.6, 102.3, 100.1, 80.3, 74.9, 74.0, 73.5, 68.5, 68.4, 66.8, 56.6, 44.4, 41.3, 38.1, 21.2, 20.5, 14.1.ESI-HRMS (ESI +) m / z calcd. for [M+Na] + , 776.17972; found, 776.17989.

[0312] Synthesis Example 12 Synthesis of ETP-βGlcNAc ETP-βGlcNAc, a compound of the present invention, was synthesized according to the following scheme.

[0313] Compound 11 (214 mg, 0.284 mmol), compound 10 (161 mg, 0.284 mmol), and triethylamine (2.0 mL) were dissolved in 5.0 mL of N,N-dimethylformamide, and the solution was stirred at room temperature under an argon atmosphere for 12 hours. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (CH ２ Cl ２ This was purified using a gradient of 100:0 to 0:100 NaOH (NaOH:MeOH) to obtain an acetylated glycoside adduct of etoposide. This was dissolved in 10.0 mL of methanol, and NaOMe (107 mg, 1.98 mmol) was added and stirred at room temperature for 1 hour. Amberlite IR120 (300 mg) was added, and after stirring for 10 minutes, the mixture was removed by suction filtration and the solvent was distilled off under reduced pressure. The product was purified by HPLC to obtain a white solid ETP-βGlcNAc (192 mg, 0.182 mmol) in a yield of 64.1%. The target product was １ H NMR, １３ The product was identified by CNMR and ESI-HRMS.

[0314] 1 H NMR (400 MHz, MeOD): δ7.40-7.24 (m, 2H), 7.15 (s, 1H), 7.01-6.91 (m, 2H), 6.61-6.57 (m, 3H), 5.92 (d, J = 6.8 Hz, 2H), 5.372 (m, 1H, overlaps with HOD), 4.84 (s, 1H), 4.73 (d, J = 4.8 Hz, 1H), 4.50-4.47 (m, 1H), 4.39 (s, 1H), 4.35-4.25 (m, 1H), 4.15-4.05 (m, 1H), 3.99-3.89 (m, 4H), 3.76-3.71 (m, 9H), 3.66-3.45 (m, 10H), 3.34-3.20 (m, 5H), 3.11-2.97 (m, 5H), 2.88 (s, 1H), 2.03 (s, 3H), 1.99 (s, 2H), 1.30 (d, J = 5.2 Hz, 1H). 13C NMR (101 MHz, MeOD): δ180.8, 173.1, 159.1, 158.7, 158.0, 157.1, 155.4, 155.2, 153.3, 148.6, 147.2, 140.7, 132.4, 131.3, 131.2, 130.2, 129.9, 129.6, 129.1, 117.4, 116.8, 114.2, 109.4, 107.9, 105.4, 102.1, 101.7, 99.8, 80.6, 77.2, 75.5, 75.0, 73.8, 70.9, 69.2, 68.2, 67.1, 66.5, 61.6, 56.4, 55.9, 55.8, 45.6, 44.1, 39.7, 34.7, 34.5, 22.1, 19.8, 0.0. [M+Na] + , 1078.36389; found, 1078.36515.

[0315] Synthesis of Compound 12 Compound 12 was synthesized according to the following scheme.

[0316] Compound 6 (5.05 g, 11.2 mmol) was dissolved in 10.0 mL of methanol, and NaOMe (2.00 g, 37.0 mmol) was added and stirred at room temperature for 1 hour. Amberlite IR120 (2.00 g) was added, and after stirring for 10 minutes, the mixture was removed by suction filtration, and the solvent was distilled off under reduced pressure. Silica gel column chromatography (CH ２ Cl ２The deprotected compound of Compound 1 was obtained by purifying the compound using a gradient of AcOEt:MeOH (100:0 to 0:100). This compound was dissolved in 15.0 mL of pyridine, and allyl chloroformate (13.0 g, 108 mmol) was added while cooling in an ice bath, followed by stirring at room temperature for 20 hours. Dichloromethane was added, and the mixture was washed with saturated aqueous sodium bicarbonate, purified water, and saturated saline, and the organic phase was extracted. The organic phase was dried over sodium sulfate, filtered, and the solvent was removed under reduced pressure. The mixture was purified by silica gel column chromatography (AcOEt:hexane (0:100 to 100:0) gradient) to obtain Compound 12 (144 mg, 0.249 mmol) as a colorless solid in a yield of 2.2%. The target product obtained was １ H NMR, １３ The product was identified by CNMR and ESI-HRMS.

[0317] 1 H NMR (400 MHz, CD2Cl2): δ8.29 (d, J = 9.2 Hz, 2H), 7.41 (d, J = 9.2 Hz, 4H), 7.10 (d, J = 8.8 Hz, 2H), 6.33 (d, J = 8.4 Hz, 1H), 6.01-5.91 (m, 3H), 5.59 (d, J = 8.4 Hz, 1H), 5.52 (t, J = 9.2 Hz, 1H), 5.40-5.26 (m, 8H), 5.01 (t, J = 10 Hz, 1H), 4.67-4.46 (m, 4H), 4.62 (d, J = 5.6 Hz, 1H), 4.43-4.39 (m, 1H), 4.35-4.31 (m, 1H), 4.15-4.00 (m, 2H),1.94 (s, 3H). 13C NMR (101 MHz, CD2Cl2): δ171.1, 157.8, 156.0, 155.1, 154.9, 154.3, 152.8, 145.8, 132.0, 131.7, 131.7, 130.8, 129.5, 125.6, 122.3, 119.4, 119.2, 119.0, 117.4, 98.3, 75.9, 73.0, 71.8, 70.9, 69.6, 69.4, 69.1, 65.9, 55.5, 23.5. ESI-HRMS (ESI +) m / z calcd. for [M+Na] + , 767.19062; found, 767.19413.

[0318] Synthesis of Compound 13 Compound 13 was synthesized according to the following scheme.

[0319] Compound 12 (144 mg, 0,249 mmol), NaBH ４ (7.50 mg, 0.198 mmol) was dissolved in a mixed solvent of 3.0 mL of dichloromethane and 3.0 mL of methanol, and stirred at room temperature for 30 minutes. Purified water was added, and the mixture was washed with dichloromethane to extract the target product. The organic phase was washed with saturated brine and then dried over sodium sulfate. After filtration, the solvent was distilled off under reduced pressure to obtain a white solid intermediate. This was dissolved in 20.0 mL of dichloromethane, and p-nitrophenyl chloroformate (150 mg, 0.750 mmol) and pyridine (60.3 μL) were added and stirred for 1 hour. The solvent was distilled off under reduced pressure, and the mixture was purified by silica gel column chromatography (AcOEt:hexane = 0:100 to 100:0 gradient) to obtain compound 13 (89.3 mg, 0.120 mmol) as a white solid in a yield of 47.9%. The target product obtained was １ H NMR, １３ The product was identified by CNMR and ESI-HRMS.

[0320] 1H NMR (400 MHz, CD2Cl2): δ9.874 (s, 1H), 7. 88 (d, J = 8.8 Hz, 2H), 7.22 (d, J = 8.8 Hz, 2H), 5.99-5.92 (m, 3H), 5.66 (d, J = 8.4 Hz, 2H), 5.41-5.24 (m, 9H), 5.04-4.99 (m, 1H), 4.64-4.63 (m, 4H), 4.59 (d, J = 5.6 Hz, 1H), 4.40-4.30 (m, 1H), 4.30-4.20 (m, 2H), 1.92 (s, 3H). 13 C NMR (101 MHz, CD2Cl2): δ192.9, 173.8, 163.2, 156.1, 156.1, 155.5, 133.1, 133.1, 133.0, 133.0, 132.9, 119.4, 119.1, 119.0, 117.9, 98.6, 77.4, 74.0, 72.8, 70.3, 70.0, 69.8, 66.8, 55.7, 23.0. ESI-HRMS (ESI +) m / z calcd. for [M+Na] + , 600.16876; found, 600.16612.

[0321] Synthesis Example 13 Synthesis of PTX-βGlcNAc PTX-βGlcNAc, a compound of the present invention, was synthesized according to the following scheme.

[0322] Compound 13 (67.2 mg, 0.0900 mmol), paclitaxel (100 mg, 0.117 mmol), and 4-dimethylaminopyridine (DMAP) (14.3 mg, 0.117 mmol) were dissolved in 5.0 mL of dichloromethane and stirred under an argon atmosphere at room temperature for 15 hours. The solvent was removed by distillation under reduced pressure, and the mixture was purified by silica gel column chromatography (AcOEt:hexane = 0:100 to 100:0 gradient) to obtain an Alloc-protected PTX-βGlcNA. This was dissolved in 8.0 mL of tetrahydrofuran, and HCOOH (8.1 μL) and triethylamine (45 μL) were added. The mixture was stirred at room temperature under an argon atmosphere for 10 minutes. While cooling in an ice bath, tetrakis(triphenylphosphine)palladium(0) (Pd(PPh ３ ) ４ ) (24.9 mg, 0.0216 mmol) was added, and the mixture was stirred at room temperature for 2 hours under an argon atmosphere. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (CH ２ Cl ２ The product was purified using a gradient of 100:0 to 85:15 (MeOH:COOH) to obtain PTX-βGlcNA (82.0 mg, 0.249 mmol) as a white solid in a yield of 75.3%. １ H NMR, １３ The product was identified by CNMR and ESI-HRMS.

[0323] 1H NMR (400 MHz, MeOD): δ8.14 (d, J = 8.4 Hz, 2H), 7.79 (d, J = 8.4 Hz, 2H), 7.68 (t, J = 7.6 Hz, 2H), 7.61-7.57 (m, 3H), 7.55-7.50 (m, 3H), 7.47-7.41 (m, 4H), 7.32-7.26 (m, 3H), 7.02 (d, J = 8.4 Hz, 2H), 6.48 (s, 1H), 6.10 (t, J = 8.8 Hz, 1H), 5.86 (d, J = 6.4 Hz, 1H), 5.66 (d, J = 7.2 Hz, 1H), 5.50 (d, J = 6.4 Hz, 1H), 5.16 (d, J = 4.0 Hz, 2H), 5.01 (d, J = 9.2 Hz, 2H), 4.39-4.36 (m, 1H), 4.20 (s, 2H), 3.97-3.92 (m, 2H), 3.84 (d, J = 6.8 Hz, 1H), 3.75 (dd, J = 4.8 Hz, J = 12 Hz, 1H), 3.61 (t, J = 7.6 Hz, 1H), 3.47-3.46 (m, 2H), 3.37-3.33 (m, 1H), 2.43 (s, 3H), 2.19 (s, 3H), 2.00 (s, 3H), 1.95 (s, 2H), 1.89-1.80 (m, 3H), 1.68 (s, 3H), 1.17 (s, 3H), 1.15 (s, 3H). 13C NMR (101 MHz, MeOD): δ205.2, 174.0, 171.7, 171.4, 170.5, 170.3, 167.7, 159.4, 155.8, 142.3, 138.2, 135.4, 135.0, 134.7, 133.0, 131.4, 131.2, 130.5, 130.2, 129.8, 129.6, 128.6, 117.8, 100.7, 85.9, 82.3, 79.1, 78.6, 78.3, 77.5, 76.8, 76.3, 75.8, 73.2, ESI-HRMS (ESI +) m / z calcd. for [M+Na] + , 1229.43124; found, 1229.43200.

[0324] Synthesis Example 14 Synthesis of DTX-βGlcNAc DTX-βGlcNAc, a compound of the present invention, was synthesized according to the following scheme.

[0325] Compound 13 (10.0 mg, 0.0134 mmol), docetaxel (31.0 mg, 0.0384 mmol), and 4-dimethylaminopyridine (DMAP) (15.6 mg, 0.128 mmol) were dissolved in 5.0 mL of dichloromethane and stirred under an argon atmosphere at room temperature for 15 hours. The solvent was removed by distillation under reduced pressure, and the mixture was purified by silica gel column chromatography (AcOEt:hexane = 0:100 to 90:10 gradient) to obtain an Alloc-protected DTX-βGlcNA. This was dissolved in 5.0 mL of tetrahydrofuran, and HCOOH (2.0 mL) and triethylamine (20 μL) were added. The mixture was stirred at room temperature under an argon atmosphere for 10 minutes. While cooling in an ice bath, tetrakis(triphenylphosphine)palladium(0) (Pd(PPh ３ ) ４) (5.00 mg, 0.00448 mmol) was added, and the mixture was stirred at room temperature for 2 hours under an argon atmosphere. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (CH ２ Cl ２ The resulting product was purified using a gradient of 100:0 to 85:15 (MeOH:COOH) to give DTX-βGlcNA (14.1 mg, 0.0122 mmol) as a white solid in a yield of 89.8%. １ H NMR, １３ The product was identified by CNMR and ESI-HRMS.

[0326] 1 H NMR (400 MHz, MeOD): δ8.11 (d, J = 7.2 Hz, 2H), 7.68 (d, J = 7.2 Hz, 2H), 7.59-7.55 (m, 3H), 7.40-7.39 (m, 4H), 7.35 (d, J = 8.4 Hz, 2H), 7.02 (d, J = 8.4 Hz, 2H), 6.16-6.09 (m, 1H), 5.63 (d, J = 7.2 Hz, 1H), 5.35-5.27 (m, 2H), 5.14-4.99 (m, overlaps with HOD), 4.30-4.19 (m, 3H), 3.93-3.85 (m, 3H), 3.72 (dd, J = 5.2 Hz, J = 12 Hz, 2H), 3.70-3.50 (m, 2H), 3.45-3.42 (m, 2H), 2.50-2.40 (m, 1H), 2.39 (s, 2H), 2.24-2.19 (m, 1H), 1.98 (s, 3H), 1.87 (s, 3H), 1.69 (s, 3H), 1.39 (s, 9H), 1.20-1.11 (m, 6H). 13C NMR (101 MHz, MeOD): δ211.1, 174.0, 171.6, 170.3, 167.7, 159.4, 155.8, 139.5, 139.5, 137.9, 134.6, 133.1, 133.0, 131.5, 131.2, 130.6, 130.1, 129.9, 129.7, 129.4, 128.3, 117.7, 100.8, 86.0, 82.3, 80.9, 79.2, 78.3, 77.6, 76.5, 75.8, 75.6, 73.5, 72.6, 71.9, 71.1, 62.6, 58.9, 57.4, 56.4, 47.9, 44.5, 37.5, 36.6, 28.7, 27.0, 23.3, 23.0, 21.7, 14.6, 10.5. ESI-HRMS (ESI +) m / z calcd. for [M+Na] + , 1183.44689; found, . 1183.44764.

[0327] Synthesis Example 15 Synthesis of CBZ-βGlcNAc CBZ-βGlcNAc, a compound of the present invention, was synthesized according to the following scheme.

[0328] Compound 13 (12.0 mg, 0.0161 mmol), cabazitaxel (40 mg, 0.0479 mmol), and 4-dimethylaminopyridine (DMAP) (19.6 mg, 0.160 mmol) were dissolved in 5.0 mL of dichloromethane and stirred under an argon atmosphere at room temperature for 15 hours. The solvent was removed by distillation under reduced pressure, and the mixture was purified by silica gel column chromatography (AcOEt:hexane = 0:100 to 70:30 gradient) to obtain an Alloc-protected CBZ-βGlcNA. This was dissolved in 5.0 mL of tetrahydrofuran, and HCOOH (2.0 μL) and triethylamine (20 μL) were added. The mixture was stirred at room temperature under an argon atmosphere for 10 minutes. While cooling in an ice bath, tetrakis(triphenylphosphine)palladium(0) (Pd(PPh ３ ) ４) (5.0 mg, 0.00448 mmol) was added, and the mixture was stirred at room temperature for 2 hours under an argon atmosphere. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (CH ２ Cl ２ The resulting product was purified using a gradient of 100:0 to 85:15 (MeOH:COOH) to give CBZ-βGlcNA (17.7 mg, 0.0149 mmol) as a white solid in a yield of 92.4%. １ H NMR, １３ The product was identified by CNMR and ESI-HRMS.

[0329] 1 H NMR (400 MHz, MeOD): δ8.11 (d, J = 7.6 Hz, 2H), 7.67-7.65 (m, 1H), 7.56 (t, J = 7.6 Hz, 3H), 7.50-7.41 (m, 4H), 7.03 (d, J = 8.4 Hz, 2H), 6.13-6.11 (m, 1H), 5.60 (d, J = 6.8 Hz, 1H), 5.35-5.30 (m, 1H), 5.30-5.23 (m, 3H), 5.13 (s, 2H), 5.08-5.00 (m, overlaps with HOD), 4.18 (q, J = 8.0Hz, 2H), 3.93-3.88 (m, 3H), 3.81 (d, J = 6.8 Hz, 1H), 3.72 (dd, J = 5.2 Hz, J = 12 Hz, 2H), 3.60-3.55 (m, 2H), 3.45-3.40 (m, 5H), 2.78-2.72 (m, 1H), 2.40 (s, 3H), 2.19-2.18 (m, 1H), 1.98-1.96 (m, 6H), 1.67 (s, 3H), 1.40 (s, 9H), 1.15-1.12 (m, 6H). 13C NMR (101 MHz, MeOD): δ207.6, 173.9, 171.6, 170.3, 167.6, 159.5, 155.8, 141.4, 138.7, 136.1, 134.6, 131.4, 131.2, 130.5, 129.9, 129.7, 129.5, 128.3, 117.7, 100.7, 85.6, 83.8, 82.5, 82.0, 80.9, 79.1, 79.0, 78.3, 77.4, 76.1, 75.8, 73.4, 71.9, 71.1, 62.6, 58.2, 57.5, 57.4, 57.1, 56.3, 50.1, 44.7, 36.4, 33.1, 28.7, 27.2, 23.3, 23.0, 21.9, 15.0, 10.9. ESI-HRMS (ESI +) m / z calcd. for [M+Na] + , 1211.47819; found, . 1211.47639.

[0330] Synthesis of Compound 14 Compound 14 was synthesized according to the following scheme.

[0331] 4-(((tert-Butyldimethylsilyl)oxy)methyl)aniline (1.00 g, 4.22 mmol), (((9H-fluoren-9-yl)methoxy)carbonyl)-L-phenylalanine (1.63 g, 4.21 mmol), HATU (2.4 g, 6.31 mmol), and N,N-diisopropylethylamine (DIEA) (2.93 mL) were dissolved in 10.0 mL of N,N-dimethylformamide, and the mixture was stirred at room temperature for 3 hours. Dichloromethane was added, and the mixture was washed with purified water to extract the organic phase. The organic phase was washed with saturated brine and then dried over sodium sulfate. After filtration, the solvent was evaporated under reduced pressure. 10.0 mL of N,N-dimethylformamide in which 20% piperidine (PPD) had been dissolved was added, and the mixture was stirred at room temperature for 20 minutes. Dichloromethane was added, and the mixture was washed with purified water to extract the organic phase. The organic phase was washed with saturated saline, dried over sodium sulfate, and filtered, after which the solvent was distilled off under reduced pressure. ２ Cl ２The resulting mixture was purified using a gradient of 100:0 to 90:10 (MeOH:MeOH) to give Compound 9 (500 mg, 1.30 mmol) in a yield of 30.9%. The target product was identified by ESI-HRMS. ESI-HRMS (ESI +) m / z calcd. for [M+Na] + , 407.21307; found, 407.21233.

[0332] Synthesis of Compound 15 Compound 15 was synthesized according to the following scheme.

[0333] Compound 14 (500 mg, 1.30 mmol), N-(tert-butoxycarbonyl)-O-(tert-butyl)-L-threonine (537 mg, 1.95 mmol), HATU (742 mg, 1.95 mmol), and N,N-diisopropylethylamine (DIEA) (906 μL) were dissolved in 15.0 mL of N,N-dimethylformamide and stirred at room temperature for 2 hours. Dichloromethane was added, and the mixture was washed with purified water to extract the organic phase. The organic phase was washed with saturated saline and then dried over sodium sulfate. After filtration, the solvent was evaporated under reduced pressure. Silica gel column chromatography (CH ２ Cl ２ This was purified by a gradient of 100:0 to 95:5 (MeOH:MeOH) to give an intermediate compound, which was dissolved in a mixed solvent of 8.0 mL of methanol and 2.0 mL of water, and added with KHSO ４ (931 mg, 6.83 mmol) was added and stirred at room temperature for 30 minutes. Ethyl acetate was added, washed with purified water, and the organic phase was extracted. The organic phase was washed with saturated brine, dried over sodium sulfate, and filtered, and the solvent was distilled off under reduced pressure. Purification was performed by silica gel column chromatography (AcOEt:hexane = 0:100 to 50:50 gradient) to obtain compound 10 (406 mg, 0.770 mmol) as a white solid in a yield of 56.2%. The target product obtained was １ The product was identified by HNMR and ESI-HRMS.

[0334] 1H NMR (400 MHz, CD2Cl2): δ8.33 (s, 1H), 7.45 (d, J = 8.0 Hz, 2H), 7.26-7.15 (m, 7H), 6.50 (d, J = 9.2 Hz, 1H), 5.52 (d, J = 5.6 Hz, 1H), 4.83-4.75 (m, 1H), 4.53 (s, 2H), 4.05-3.96 (m, 3H), 3.62 (dd, J = 4.8 Hz, J = 13.6 Hz, 1H), 2.95 (dd, J = 5.6 Hz, J = 13.6 Hz, 1H), 1.31 (s, 9H), 1.02-0.99 (m, 12H). ESI-HRMS (ESI +) m / z calcd. for [M+Na] + , 550.28876; found, 550.28779.

[0335] Synthesis of Compound 16 Compound 16 was synthesized according to the following scheme.

[0336] Compound 15 (406 mg, 0.770 mmol), PBr ３ (72 μL) was dissolved in 10.0 mL of dichloromethane and stirred at room temperature for 1 hour. A saturated aqueous solution of sodium bicarbonate was added, and the organic phase was extracted. The organic phase was washed with purified water and saturated brine, then dried over sodium sulfate, and filtered, after which the solvent was distilled off under reduced pressure. Purification was carried out by silica gel column chromatography (AcOEt:hexane = 0:100 to 70:30 gradient) to obtain compound 11 (180 mg, 0.307 mmol) as a white solid in a yield of 39.9%. The target product obtained was １ The product was identified by HNMR and ESI-HRMS.

[0337] 1H NMR (400 MHz, CD2Cl2): δ8.54 (s, 1H), 7.43 (d, J = 8.0 Hz, 2H), 7.21-7.10 (m, 7H), 6.70 (d, J = 8.8 Hz, 1H), 5.53 (d, J = 5.6 Hz, 1H), 4.80-4.75 (m, 1H), 4.38 (s, 1H), 4.02-3.97 (m, 2H), 3.27 (dd, J = 5.2 Hz, J = 13.6 Hz, 1H), 2.95 (dd, J = 6.0 Hz, J = 13.6 Hz, 1H), 3.24-3.15 (m, 3H), 3.09-3.04 (m, 1H), 2.10-1.90 (m, 2H), 1.33 (t, J = 7.6 Hz, 3H), 1.18 (d, J = 6.4 Hz, 3H), 1.30 (s, 9H), 1.00 (s, 12H). ESI-HRMS (ESI +) m / z calcd. for [M+Na] + , 612.20436; found, 612.20521.

[0338] Synthesis Example 16 Synthesis of TF-SN38 TF-SN38, a compound of the present invention, was synthesized according to the following scheme.

[0339] Compound 16 (180 mg, 0.307 mmol), 7-ethyl-10-hydroxycamptothecin (SN38) (359 mg, 0.916 mmol), Cs ２ CO ３ (299 mg, 0.918 mmol) was dissolved in 15.0 mL of N,N-dimethylformamide and stirred at room temperature for 1 hour. Dichloromethane was added, and the mixture was washed with purified water and saturated brine, dried over sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. ２ Cl ２The resulting mixture was purified by a gradient of 100:0 to 95:5 (MeOH: 100:0 to 95:5) to obtain an intermediate compound. A mixture of 7.5 mL of trifluoroacetic acid, 3.5 mL of dichloromethane, and 125 μL of triisopropylsilane (TIS) was added, and the mixture was stirred at room temperature for 5 hours. After the solvent was distilled off under reduced pressure, the mixture was purified by silica gel column chromatography (CH ２ Cl ２ The crude target product was obtained by HPLC purification, and a pale yellow solid TF-SN38 (31.0 mg, 0.0416 mmol) was obtained in a yield of 13.6%. １ H NMR, １３ The product was identified by CNMR and ESI-HRMS.

[0340] 1 H NMR (400 MHz, MeOD): δ8.03 (d, J = 9.2 Hz, 1H), 7.60-7.46 (m, 6H), 7.43 (d, J = 2.8 Hz, 1H), 7.31-7.20 (m, 5H), 5.58 (d, J = 16 Hz, 1H), 5.38 (d, J = 16 Hz, 1H), 5.23 (d, J = 8.4 Hz, 4H), 4.80-4.77 (m, 1H), 3.98-3.94 (m, 1H), 3.27 (d, J = 4.8 Hz, 1H), 3.24-3.15 (m, 3H), 3.09-3.04 (m, 1H), 2.10-1.90 (m, 2H), 1.33 (t, J = 7.6 Hz, 3H), 1.18 (d, J = 6.4 Hz, 3H), 1.03 (t, J = 7.6 Hz, 3H). 13C NMR (101 MHz, MeOD): δ172.65, 172.54, 169.70, 157.12, 156.80, 150.03, 149.58, 146.24, 144.39, 143.82, 138.43, 137.13, 131.50, 131.41, 129.22, 129.70, 128.35, 128.06, 127.73, 126.40, 122.62, 119.34, 118.21, 103.65, 95.99, 72.37, 69.54, 67.60, 65.20, 60.08, 54.12, 49.50, 37.94, 30.16, 22.20, 19.83, 13.42, 7.73. ESI-HRMS (ESI +) m / z calcd. for [M+Na] + , 768.30038; found, 768.30030.

[0341] Synthesis of Compound 17 Compound 17 was synthesized according to the following scheme.

[0342] 4-(((tert-Butyldimethylsilyl)oxy)methyl)aniline (2.00 g, 8.44 mmol), (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanine (2.63 g, 8.45 mmol), HATU (4.81 g, 12.6 mmol), and N,N-diisopropylethylamine (DIEA) (5.87 mL) were dissolved in 10.0 mL of N,N-dimethylformamide and stirred at room temperature for 3 hours. Ethyl acetate was added, and the mixture was washed with purified water to extract the organic phase. The organic phase was washed with saturated saline and then dried over sodium sulfate. After filtration, the solvent was evaporated under reduced pressure. 20.0 mL of N,N-dimethylformamide in which 20% piperidine (PPD) had been dissolved was added, and the mixture was stirred at room temperature for 20 minutes. Ethyl acetate was added, and the mixture was washed with purified water to extract the organic phase. The organic phase was washed with saturated saline and then dried over sodium sulfate. After filtration, the solvent was evaporated under reduced pressure. Silica gel column chromatography (CH ２ Cl ２The resulting mixture was purified using a gradient of 100:0 to 90:10 NaCl / MeOH to give compound 12 (2.16 g, 5.56 mmol) in a yield of 65.9%. The target compound was identified by ESI-HRMS. ESI-HRMS (ESI +) m / z calcd. for [M+Na] + 331.18123; found 331.18179.

[0343] Synthesis of Compound 18

[0344] Compound 17 (2.00 g, 6.49 mmol), N ２ -(tert-butoxycarbonyl)-N ５ N,N-trityl-L-glutamine (4.00 g, 8.19 mmol), HATU (3.70 mg, 9.74 mmol), and N,N-diisopropylethylamine (DIEA) (4.52 mL) were dissolved in 15.0 mL of N,N-dimethylformamide and stirred at room temperature for 2.5 hours. Ethyl acetate was added, and the mixture was washed with purified water to extract the organic phase. The organic phase was washed with saturated brine, dried over sodium sulfate, and filtered, followed by distillation under reduced pressure to remove the solvent. The intermediate compound was purified by silica gel column chromatography (AcOEt:hexane = 0:100 to 100:0 gradient) to obtain the intermediate compound. This intermediate compound was dissolved in a mixed solvent of 20.0 mL of methanol and 5.0 mL of water, and the resulting mixture was diluted with KHSO. ４ (4.41 g, 32.4 mmol) was added and stirred at room temperature for 1 hour. After distilling off under reduced pressure and removing half of the methanol, dichloromethane was added and washed with purified water to extract the organic phase. The organic phase was washed with saturated saline and then dried over sodium sulfate, and after filtration, the solvent was distilled off under reduced pressure. Silica gel column chromatography (CH ２ Cl ２ The resulting mixture was purified using a gradient of 100:0 to 95:5 NaCl, MeOH, to give compound 18 (1.36 g, 2.05 mmol) in a 31.5% yield. The target product was identified by ESI-HRMS. ESI-HRMS (ESI +) m / z calcd. for [M+Na] + 687.31531; found 687.31599.

[0345] Synthesis of Compound 19 Compound 19 was synthesized according to the following scheme.

[0346] Compound 18 (340 mg, 0.512 mmol), PBr ３ (48 μL) was dissolved in 10.0 mL of dichloromethane and stirred at room temperature for 1 hour. A saturated aqueous solution of sodium bicarbonate was added, and the organic phase was extracted. The organic phase was washed with purified water and saturated brine, dried over sodium sulfate, filtered, and the solvent was evaporated under reduced pressure to obtain compound 19 (276 mg, 0.380 mmol) as a white solid in an 86.1% yield.

[0347] Synthesis Example 17 Synthesis of QA-SN38 QA-SN38, a compound of the present invention, was synthesized according to the following scheme.

[0348] Compound 19 (200 mg, 0.319 mmol), 7-ethyl-10-hydroxycamptothecin (SN38) (100 mg, 0.255 mmol), Cs ２ CO ３ (312 mg, 0.958 mmol) was dissolved in 15.0 mL of N,N-dimethylformamide and stirred at room temperature for 2 hours. Dichloromethane was added, and the mixture was washed with purified water and saturated brine, dried over sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. ２ Cl ２ The mixture was purified with a gradient of 100:0 to 80:20 (MeOH: 100:0 to 80:20) to obtain an intermediate compound. A mixed solvent of 1.5 mL of trifluoroacetic acid, 1.0 mL of dichloromethane, and triisopropylsilane (TIS) (72 μL) was added, and the mixture was stirred at room temperature for 5 hours. After the solvent was distilled off under reduced pressure, the mixture was purified by HPLC to obtain a pale yellow solid, QA-SN38 (5.2 mg, 0.00747 mmol), in a yield of 2.3%. The obtained target product was １ H NMR, １３ The product was identified by CNMR and ESI-HRMS.

[0349] 1H NMR (400 MHz, MeOD): δ8.04 (d, J = 9.2 Hz, 1H), 7.64-7.60 (m, 3H), 7.54-7.50 (m, 3H), 7.46 (d, J = 9.2 Hz, 1H), 5.58 (d, J = 14 Hz, 1H), 5.38 (d, J = 16.4 Hz, 1H), 5.25 (d, J = 10.8 Hz, 1H), 4.57-4.52 (m, 1H), 4.00 (t, J = 6.4 Hz, 1H), 3.21-3.17 (m, 2H), 2.54 (t, J = 7.2 Hz, 2H), 2.19-2.14 (m, 2H), 2.00-1.94 (m, 2H), 1.51 (d, J = 7.2 Hz, 3H), 1.35 (t, J = 7.6 Hz, 3H), 1.03 (t, J = 7.6 Hz, 3H). 13 C NMR (101 MHz, MeOD): δ177.3, 175.0, 173.0, 169.6, 159.2, 152.8, 150.6, 148.0, 146.1, 146.0, 139.5, 134.0, 132.1, 129.5, 129.4, 129.3, 124.5, 121.3, 119.8, 104.6, 99.1, 74.3, 71.1, 66.7, 53.7, 51.3, 50.8, 32.0, 31.4, 28.2, 23.9, 18.2, 13.9, 8.2. ESI-HRMS (ESI +) m / z calcd. for [M+Na] + , 719.27998; found, 719.27903.

[0350] Example 21 Cytotoxicity Test Results Using JFCR39 Cell Panel The cytotoxicity test results for the JFCR39 cell panel (7 lung cancer lines (including adenocarcinoma, squamous cell carcinoma, small cell carcinoma, and large cell carcinoma), 6 gastric cancer lines, 5 colon cancer lines, 5 ovarian cancer lines, 6 brain tumor lines, 5 breast cancer lines, 2 renal cancer lines, 2 prostate cancer lines, and 1 melanoma line) were compared with the existing drugs SN38, CPT-11, nogitecan, exatecan, etoposide, doxorubicin, epirubicin, melphalan, MMAE, MMAF, paclitaxel, docetaxel, and cabazitaxel, as well as the novel prodrug of the present invention. The cell proliferation inhibitory effects of SN38-βGal, SN38-βGlcNAc, SN38-αMan, KK-SN38, QA-SN38, TF-SN38, NGT-βGlcNAc, EXT-βGlcNAc, ETP-βGlcNAc, DOX-βGlcNAc, EPI-βGlcNAc, Melphalan (Mel)-βGlcNAc, MMAE-βGlcNAc, MMAF-βGlcNAc, PTX-βGlcNAc, DTX-βGlcNAc, and CBZ-βGlcNAc were evaluated. As shown in the table in Figure 38, these prodrugs were shown to be able to inhibit the proliferation of various cancer cell lines (Figures 39-53). In lung cancer, pharmacological efficacy was confirmed against adenocarcinoma, squamous cell carcinoma, small cell carcinoma, and large cell carcinoma.

[0351] The specific test procedure is as follows: Each cancer cell was plated on a 96-well plate, and the next day, a drug solution was added in a dilution series. After culturing for 2 days (48 hours), cell proliferation was measured by colorimetry using sulforhodamine B.

[0352] The table in Figure 38 shows the GI in each cancer cell. ５０ Here, -4.00 is the GI ５０ The value is 10 －４ Indicates that this is the case.

[0353] Figures 39 to 53 show the inhibition of cell proliferation of each cancer cell line by SN38-αMan, KK-SN38, QA-SN38, TF-SN38, NGT-βGlcNAc, EXT-βGlcNAc, ETP-βGlcNAc, DOX-βGlcNAc, EPI-βGlcNAc, Melphalan-βGlcNAc, MMAE-βGlcNAc, MMAF-βGlcNAc, PTX-βGlcNAc, DTX-βGlcNAc, and CBZ-βGlcNAc, respectively.

[0354] Example 22: Therapeutic Experiments with Each Prodrug in a Peritoneal Dissemination Model Therapeutic efficacy of the various prodrug-type anticancer agents (DOX-βGlcNAc, Mel-βGlcNAc, MMAE-βGlcNAc, PTX-βGlcNAc, ETP-βGlcNAc, and SN38-βGlcNAc) was examined using a peritoneal dissemination model. The tumor-suppressing effect was evaluated compared to the vehicle group (DMSO administration). Each group consisted of n=5, and evaluation was performed on a BALB / cAJcl-nu / nu luciferase-expressing A549 cell peritoneal dissemination mouse model. The administration route was intraperitoneal (i.p.), and administration was performed according to the administration schedules shown in Figures 54-a to 54-f. Tumor burden was evaluated by luciferin / luciferase luminescence. As a result, tumor growth suppression effects were confirmed in the prodrug administration group under each administration condition compared to the vehicle group. The above results confirmed the therapeutic effect of the developed prodrug in cancer model mice.

[0355] The specific procedure for the therapeutic experiment is as follows: (1) BALB / cAJcl-nu / nu (7 weeks old) was inoculated with 1 x 10 luciferase-expressing A549 cells. ６ Cells (200 μL) were administered intraperitoneally. (2) One week later, the formation of peritoneal dissemination was confirmed by luciferin / luciferase chemiluminescence, and drug administration was initiated. Luciferin was administered intraperitoneally at 3 mg (200 μL). After anesthesia with isoflurane, luminescence intensity was measured 15 minutes after administration using an in vivo imaging device. Drugs were prepared by dissolving 100 mM DMSO stock in PBS, and administered in a total volume of 100 μL.

Claims

1. A compound represented by the following general formula (I) or a pharma- ceutical acceptable salt thereof: １ ) ｎ (I) (Wherein, Y is -OL １ (1) or -NH-L ２ (2) is selected from L １ is a partial structure of a saccharide, where the partial structure of a saccharide is a structure obtained by removing one hydroxyl group from a saccharide, and L ２ is a monovalent substituent containing an amino acid residue or an oligopeptide residue; T is a linker or a bond; X １ is at least one selected from the group consisting of: (1) a binding residue of a biologically active compound having one or more functional groups selected from the group consisting of an aliphatic hydroxyl group, an aromatic hydroxyl group, an amino group, and a carboxyl group; (2) an antibody; and (3) a ligand selected from a small molecule, a peptide, an aptamer, or a hormone-drug conjugate, with the proviso that X is at least one selected from the group consisting of: １ at least one of the following is a binding residue of the biologically active compound; the biologically active compound is an anti-cancer drug; and n is an integer of 1 or more.

2. The compound of claim 1, or a pharma- ceutically acceptable salt thereof, wherein Y is selected from the following groups:

3. The compound according to claim 1, or a pharma- ceutically acceptable salt thereof, wherein the linker is selected from the group consisting of the following structures (a) to (f) and combinations of two or more thereof: (In the formula, R １ ~R ４ may be the same or different, and each independently represents a hydrogen atom, a methyl group, a methoxy group, -COOMe, -NHMe, F, Cl, Br, or NO ２ (Me represents a methyl group); R ５ is hydrogen or an alkyl group having 1 to 3 carbon atoms; R ６ is independently, at each occurrence, hydrogen or an alkyl group having 1 to 3 carbon atoms; １ ~R ４ At least one of may be a linking group selected from: Here, R １ ~R ４ When at least one of is a linking group, the linker is also connected to X via the linking group. １ (ii) R １ ~R ４ At least one of may be a linker having a binding site that can be used as a point of attachment to an antibody or ligand: * represents X １ However, when T has a structure that is a combination of two or more of the formulas (a) to (f), X １ * for one or more linkers other than the linker bonded to X may represent the direction of bonding to the linker; １ is an antibody or a ligand, the linkers of formulas (a)-(f), the linking groups of a1-a3 can be attached in the direction indicated by * to a linker having a binding site that can be used as a point of attachment to the antibody or ligand.

4. The compound of claim 1, or a pharma- ceutically acceptable salt thereof, wherein the biologically active compound is selected from the following:

5. The compound of claim 1, or a pharma- ceutically acceptable salt thereof, wherein the biologically active compound is SN38, nogitecan, exatecan, etoposide, doxoribicin, epirubicin, melphalan, MMAE, MMAF, paclitaxel, docetaxel, or cabazitaxel.

6. The compound of claim 2, or a pharma- ceutically acceptable salt thereof, wherein the biologically active compound is SN38, nogitecan, exatecan, etoposide, doxoribicin, epirubicin, melphalan, MMAE, MMAF, paclitaxel, docetaxel, or cabazitaxel.

7. A prodrug-type anticancer drug comprising the compound according to any one of claims 1 to 6 or a pharma- ceutically acceptable salt thereof.

8. A prodrug-type anticancer drug which acts selectively on cells by virtue of a cancer cell-specific enzyme activity, comprising the compound according to any one of claims 1 to 6 or a pharma-ceutical acceptable salt thereof.

9. The prodrug-type anticancer drug according to claim 8, wherein the enzyme is a peptidase or a glycosidase.

10. A fluorescent probe for detecting lung cancer, comprising a compound represented by the following general formula (II) or a salt thereof: (In the formula, R １ R represents, when present, the same or different monovalent substituents present on the benzene ring; ２ and R ３ each independently represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom; R ４ and R ５ each independently represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom; R ６ represents a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, or a fluorinated alkyl group having 1 to 5 carbon atoms; R ７ and R ８ each independently, if present, represents an alkyl or aryl group having 1 to 6 carbon atoms, where, when X is an oxygen atom, R ７ and R ８ is absent; X represents an oxygen atom, a silicon atom, or a carbon atom; n is an integer of 1 to 3; and L is a group represented by the following formula (1b):

11. The fluorescent probe according to claim 10, wherein X is an oxygen atom.

12. R ６ The fluorescent probe according to claim 10 or 11, wherein is a fluorinated alkyl group having 1 to 5 carbon atoms.

13. A companion diagnostic for diagnosing the need for cancer treatment with a prodrug-type anticancer drug comprising a compound represented by the following general formula (I) or a pharma- ceutical acceptable salt thereof, the companion diagnostic comprising a compound represented by the following general formula (II) or a salt thereof: Y-T-(X １ ) ｎ (I) wherein Y is selected from any of the following groups: T is a linker or bond: X １ is at least one selected from the group consisting of: (1) a binding residue of a biologically active compound having one or more functional groups selected from the group consisting of an aliphatic hydroxyl group, an aromatic hydroxyl group, an amino group, and a carboxyl group; (2) an antibody; and (3) a ligand selected from a small molecule, a peptide, an aptamer, or a hormone-drug conjugate, with the proviso that X is at least one selected from the group consisting of: １ at least one of the following is a binding residue of the biologically active compound; the biologically active compound is an anti-cancer drug; and n is an integer of 1 or more. (In the formula, R １ R represents, when present, the same or different monovalent substituents present on the benzene ring; ２ and R ３ each independently represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom; R ４ and R ５ each independently represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom; R ６ represents a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, or a fluorinated alkyl group having 1 to 5 carbon atoms; R ７ and R ８ each independently, if present, represents an alkyl or aryl group having 1 to 6 carbon atoms, where, when X is an oxygen atom, R ７ and R ８ is absent; X represents an oxygen atom, a silicon atom, or a carbon atom; n is an integer of 1 to 3; and L is selected from any of the following groups:

14. A pharmaceutical composition for treating or preventing cancer, comprising a compound according to any one of claims 1 to 6 or a pharma- ceutically acceptable salt thereof.

15. A pharmaceutical composition for treating or preventing cancer, comprising a compound represented by general formula (I) or a pharma- ceutically acceptable salt, hydrate, or solvate thereof, and, in addition, another anticancer agent.

16. The pharmaceutical composition according to claim 15, wherein the other anticancer drug is selected from methotrexate, doxorubicin, cisplatin, or an anti-PD-1 antibody.

17. A method for treating a cancer patient, comprising the step of administering to the patient a therapeutically effective amount of a prodrug-type anticancer agent according to any one of claims 7 to 9.

18. The method of treatment according to claim 17, wherein the prodrug-type anticancer drug acts cell-selectively by virtue of an enzyme activity specific to cancer cells, the cancer being characterized by a higher expression level of the enzyme.

19. The method of treatment according to claim 17, wherein the prodrug-type anticancer drug acts cell-selectively by virtue of an enzyme activity specific to cancer tissue, the cancer being characterized by a higher expression level of the enzyme.

20. The therapeutic method according to claim 18, wherein the prodrug-type anticancer drug releases the anticancer drug molecule by cleavage of the enzyme recognition site and / or the linker site due to an enzyme activity specific to cancer cells.

21. The method of treatment according to claim 19, wherein the prodrug-type anticancer drug releases the anticancer drug molecule by cleavage of the enzyme recognition site and / or the linker site due to an enzyme activity specific to cancer tissue.

22. The method of treatment of claim 20, comprising using one or more enzyme diagnostic agents selected from visualization agents or enzyme activity reporters to identify the level of enzymatic activity of said enzyme in a patient's biological sample and to define groups likely to respond to treatment as a function of said enzymatic activity.

23. The method of treatment according to claim 20 or 22, comprising using the enzyme diagnostic to stratify a set of patients and define a subset of patients likely to respond to the prodrug-type anticancer drug.

24. Use of an enzyme recognition site-linker binding site represented by general formula (III), wherein the enzyme recognition site has an enzymatic activity specific to a cancer cell, for activating an antibody or ligand (such as a small molecule, peptide, aptamer or hormone drug conjugate) having a payload that is released by cleavage of the binding site by the enzymatic activity specific to the enzyme recognition site-linker binding site. Y-T1 (III) (wherein Y is -OL １ (1) or -NH-L ２ (2) is selected from L １ is a partial structure of a saccharide, where the partial structure of a saccharide is a structure obtained by removing one hydroxyl group from a saccharide, and L ２ is a monovalent substituent comprising an amino acid residue or an oligopeptide residue; T1 is a linker).

25. Use of an enzyme recognition site-linker binding site represented by general formula (III), wherein the enzyme recognition site has an enzymatic activity specific for a cancer cell, to prevent activation of a prodrug or binding of an antibody or a ligand until the enzyme recognition site-linker binding site is cleaved by the enzymatic activity specific for the binding site.

26. Use of an enzyme recognition site-linker binding site represented by general formula (III), wherein the enzyme recognition site has an enzymatic activity specific to a cancer cell, for activating an antibody or ligand having a payload that is released or activated by cleavage of the enzyme recognition site-linker binding site by an enzymatic activity specific to the binding site in a target cell or tissue that overexpresses a cancer gene.

27. Use of an enzyme recognition site-linker binding site represented by general formula (III), wherein the enzyme recognition site has an enzymatic activity specific to cancer cells, for targeting cancer.

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