Boron Neutron Capture Therapy (BNCT) Probe

Abstract

[Problem] To provide a novel compound promising as a probe for a boron neutron capture therapy (BNCT). [Solution] A compound represented by general formula (I) below or a salt thereof.

Description

[Technical field]

[0001] The present invention relates to a novel compound that is promising as a probe for boron neutron capture therapy (BNCT), and a pharmaceutical composition for use in boron neutron capture therapy using said compound. [Background technology]

[0002] It is one of the boron isotopes 10 It is known that B emits Li nuclei and alpha rays when it captures neutrons. 10 A treatment that utilizes the properties of B is called BNCT (Boron Neutron Capture Therapy). In BNCT, first, cancer cells are 10 By accumulating B and irradiating it with neutrons, a neutron capture reaction is induced (see Figure 1).

[0003] Specifically, in BNCT, 10 B reacts with neutrons to produce alpha rays and Li nuclei, both of which have a high LET (linear energy transfer), and therefore have a higher killing power than the X-rays used in conventional radiation therapy. In addition, the alpha rays only kill cancer cells, as their range is 5-9 μm, which is shorter than the diameter of a single cell, and they do not damage adjacent normal cells. Therefore, 10 If B can be delivered, it will theoretically be possible to perform cancer-selective radiation therapy at the single-cell decomposition level. In addition, because BNCT has both high killing power and cancer cell selectivity, it can also be used for highly malignant cancer types that are resistant to radiation.

[0004] In BNCT, in order to efficiently emit alpha particles at the cancer site and minimize damage to healthy tissue, (1) high concentration 10 The two most important factors are (1) the accumulation of B (≒ several mM) and (2) high tumor selectivity. Currently, only two drugs, BSH and BPA (p-boronophenylalanine), are used in clinical research, but both drugs have high selectivity and can deliver high concentrations to cancer cells. 10However, it is difficult to accumulate B and maintain it for a sufficient period of time, which is why the excellent therapeutic effects of BNCT have not been fully realized. In other words, in order to perfect BNCT as an ideal cancer treatment technique that minimizes damage to normal tissue, it is necessary to develop a high-concentration, tumor-selective B. 10 It is necessary to develop a completely new BNCT drug that can achieve B retention. Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention aims to provide a novel compound that is promising as a probe for boron neutron capture therapy (BNCT). [Means for solving the problem]

[0006] The fluorescent probe SPiDER-βgal, developed by the inventors' laboratory, reacts with β-galactosidase to generate a quinone methide intermediate, which tags various nucleophiles within the cell, allowing only β-galactosidase-expressing cells to be fluorescently labeled with single-cell resolution.

[0007] Furthermore, the inventors' laboratory has developed a cancer-selective prodrug-type anticancer drug using quinone methide chemistry. It has been suggested that this anticancer drug uses the high reactivity of the generated azaquinone methide intermediate to consume intracellular nucleophiles, disrupting the intracellular redox balance and inducing apoptosis in cancer cells (WO 2019 / 172210, etc.).

[0008] Based on these findings, the inventors believed that if quinone methide chemistry could be adopted and covalent bond formation with intracellular nucleophiles could be used as a mechanism for intracellular retention, it would be possible to develop a BNCT drug with stronger intracellular retention, which would resolve the problem of sustained boron concentration that exists with existing BNCT drugs, and thus completed the present invention.

[0009] That is, the present invention provides: [1] A compound represented by the following general formula (I) or a salt thereof: TIFF0007628340000001.tif42153 (in the formula, X is selected from the group consisting of a fluorine atom, an ester group (-OC(=O)-R'), a carbonate group (-OCO2-R'), a carbamate group (-OCONH-R'), phosphoric acid and its ester group (-OP(=O)(-OR')(-OR''), and sulfuric acid and its ester group (-OSO2-OR'); wherein R′, R″ are each independently selected from a substituted or unsubstituted alkyl group, or a substituted or unsubstituted aryl group; Y is -NH-CO-L, -NH-L' or -OL'; where L is a substructure of an amino acid, L' is a saccharide or saccharide moiety, a saccharide having a self-cleaving linker, an amino acid or a peptide having a self-cleaving linker; R 1 and R 2 are each independently selected from a hydrogen atom or a monovalent substituent; R 3 is a hydrogen atom or 1 to 3 identical or different monovalent substituents present on a benzene ring; Z represents a single bond or a linking group: B is, 10 represents a group containing B. [2] The compound or salt thereof according to [1], wherein B is a group derived from a compound having at least one boron atom in the molecule. [3] The compound or salt thereof according to [1] or [2], wherein B is a group derived from a boron cluster. [4] The compound or salt thereof according to [3], wherein the boron cluster has a polyhedral structure. [5] The compound or salt thereof according to any one of [1] to [4], wherein B is a group derived from closododecaborate, closodocarborane, nidocarborane, bisdicarbolide metal complex, GB10, 1,2-dicarbacloso-dodecaborane, 1,7-dicarba-closo-dodecaborane, 1,12-dicarba-closo-dodecaborane, dicarba-closo-decaborane, or sulfur-substituted undecahydrododecaborate. [6] The compound or salt thereof according to any one of [1] to [5], wherein the linking group is selected from the group consisting of an alkylene group (wherein one or more -CH2- of the alkylene group may be substituted with -O-, -S-, -NH-, or -CO-), an arylene (including heteroarylene), a cycloalkylene, an alkoxyl group, a polyethylene glycol chain, and a group formed by arbitrarily bonding two or more groups selected from these groups. [7] The compound or salt thereof according to any one of [1] to [6], wherein a partial structure of the amino acid of L, together with the C=O to which it is bonded, constitutes an amino acid, an amino acid residue, a peptide, or a part of an amino acid. [8] The compound or salt thereof according to any one of [1] to [6], wherein the partial structure of the saccharide represented by L', together with the O to which it is bound, constitutes a saccharide or a part of a saccharide. [9] In the general formula (I), -Y is -C(R 1 )(R 2 ) X is bonded to the ortho-position or para-position of the benzene ring, or a salt thereof.

[10] The compound or salt thereof according to any one of [1] to [9], wherein Y has a structure selected from the following: TIFF0007628340000002.tif97159

[11] The compound or salt thereof according to any one of [1] to

[10] , wherein X is a fluorine atom or an ester group (-OC(=O)-R').

[12] R 1 and R 2 are each independently selected from a hydrogen atom or a fluorine atom,

[13] R3 and the monovalent substituent is selected from the group consisting of an alkyl group, an alkoxycarbonyl group (-C(=O)-OR'), a nitro group, an amino group, a hydroxyl group, an alkylamino group (-NHR', -NR'2), an alkoxy group (-OR'), an ester group (-O-CO-R'), an amide group (-NHCOR'), a halogen atom, a boryl group, and a cyano group (R' is a substituted or unsubstituted alkyl group, or a substituted or unsubstituted aryl group, and when there are two or more R', they may be the same or different from each other), the compound or salt thereof according to any one of [1] to

[12] .

[14] R 3

[13] The compound or salt thereof according to

[13] , wherein the monovalent substituent is an alkyl group or an alkoxy group.

[15] R 3 The compound or salt thereof according to

[13] , wherein the monovalent substituent is a halogen atom.

[16] R 3 one or more of the monovalent substituents of R is an alkyl or alkoxy group; 3 The compound or salt thereof according to any one of

[13] to

[15] , wherein one or more of the monovalent substituents represented by the formula:

[17] R 3 The compound or salt thereof according to any one of [1] to

[12] , wherein all of

[18] A pharmaceutical composition comprising the compound according to any one of [1] to

[17] or a pharma- ceutical acceptable salt thereof.

[19] The pharmaceutical composition according to

[18] , which is used in boron neutron capture therapy.

[20] The pharmaceutical composition described in

[19] , which can accumulate in cancer cells by acting selectively on cancer cells through cancer cell-specific enzymatic activity.

[21] The pharmaceutical composition described in

[20] , wherein the enzyme is a peptidase or a glycosidase.

[22] A method for diagnosing, treating, or diagnosing and treating a disease or a condition that may lead to a disease, comprising: (A) administering to a subject having or suspected of having a disease or condition a pharmaceutical composition comprising a compound according to any one of claims 1 to 17 or a pharma- ceutical acceptable salt thereof; and (B) localized to a target tissue of the subject. 10 Irradiating the B atoms with neutrons, thereby performing boron neutron capture therapy on the target tissue. The method comprising: This provides: Effect of the Invention

[0010] The present invention can provide a novel compound that is promising as a probe for boron neutron capture therapy (BNCT). [Brief description of the drawings]

[0011] [Figure 1] Conceptual diagram of BNCT (boron neutron capture therapy). [Diagram 2] 1 shows a schematic diagram of the mechanism by which the compound of the present invention is retained in cells via reaction with a cancer biomarker enzyme. [Diagram 3] The reactivity of gGlu-4OCB-FMA with the purified enzyme is shown. [Figure 4] The reactivity of EP-4OCB-FMA with purified enzymes is shown. [Diagram 5] The reactivity of EP-4OCB-MA with the purified enzyme is shown. [Figure 6] FIG. 6 shows the results of measuring cell viability after 24 hours of EP-4OCB-FMA treatment in the presence or absence of CCK8 and sitagliptin. [Figure 7] FIG. 7 shows the results of measuring cell viability following EP-4OCB-FMA treatment with or without CCK8 and sitagliptin for 3 or 24 hours. [Figure 8] 8 shows cell viability measurements upon 24 h of EP-4OCB-MA treatment with or without CCK8 and sitagliptin. Mean values ​​± standard deviation of concentrations compared to untreated are shown (n=3 biological replicates). [Figure 9] 1 shows the protocol used in the test of Example 4. [Figure 10]1 shows the results of quantifying extracellular boron concentration in Example 4. [Figure 11] 1 shows the results of quantifying intracellular boron concentration in Example 4. [Figure 12] The results of evaluation of the reactivity of the azaquinone methide intermediate and the nucleophilic group for EP-4OCB-FMA are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] In this specification, the term "halogen atom" means a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom.

[0013] In the present specification, "alkyl" may be any of linear, branched, cyclic, and aliphatic hydrocarbon groups consisting of a combination thereof. The number of carbon atoms of the alkyl group is not particularly limited, and may be, for example, 1 to 6 carbon atoms (C1-6), 1 to 10 carbon atoms (C1-10), 1 to 15 carbon atoms (C1-15), or 1 to 20 carbon atoms (C1-20). When the number of carbon atoms is specified, it means "alkyl" having the number of carbon atoms within the specified range. For example, C1-8 alkyl includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neo-pentyl, n-hexyl, isohexyl, n-heptyl, n-octyl, and the like. In the present specification, the alkyl group may have one or more optional substituents. Examples of such substituents include, but are not limited to, alkoxy groups, halogen atoms, amino groups, mono- or di-substituted amino groups, substituted silyl groups, or acyl. When an alkyl group has two or more substituents, they may be the same or different. The same applies to the alkyl moiety of other substituents containing an alkyl moiety (e.g., alkoxy groups, arylalkyl groups, etc.).

[0014] In the present specification, when a functional group is defined as "optionally substituted", the type of the substituent, the substitution position, and the number of the substituent are not particularly limited, and when it has two or more substituents, they may be the same or different. Examples of the substituent include, but are not limited to, an alkyl group, an alkoxy group, a hydroxyl group, a carboxyl group, a halogen atom, a sulfo group, an amino group, an alkoxycarbonyl group, and an oxo group. These substituents may further have a substituent. Examples of such a substituent include, but are not limited to, a halogenated alkyl group and a dialkylamino group.

[0015] In the present specification, "aryl" may be either a monocyclic or condensed polycyclic aromatic hydrocarbon group, or may be an aromatic heterocycle containing one or more heteroatoms (e.g., oxygen, nitrogen, or sulfur atoms) as ring constituent atoms. In this case, it may be called "heteroaryl" or "heteroaromatic". In either case of a monocyclic or condensed ring, aryl may be bonded at any possible position. Non-limiting examples of monocyclic aryl include phenyl group (Ph), thienyl group (2- or 3-thienyl group), pyridyl group, furyl group, thiazolyl group, oxazolyl group, pyrazolyl group, 2-pyrazinyl group, pyrimidinyl group, pyrrolyl group, imidazolyl group, pyridazinyl group, 3-isothiazolyl group, 3-isoxazolyl group, 1,2,4-oxadiazol-5-yl group, or 1,2,4-oxadiazol-3-yl group. Non-limiting examples of fused polycyclic aryls include 1-naphthyl, 2-naphthyl, 1-indenyl, 2-indenyl, 2,3-dihydroinden-1-yl, 2,3-dihydroinden-2-yl, 2-anthryl, indazolyl, quinolyl, isoquinolyl, 1,2-dihydroisoquinolyl, 1,2,3,4-tetrahydroisoquinolyl, indolyl, isoindolyl, phthalazinyl, quinoxalinyl, benzofuranyl, 2,3-dihydrobenzofuran-1-yl, 2,3-dihydrobenzofuran-2-yl, 2,3-dihydrobenzothiophen-1-yl, 2,3-dihydrobenzothiophen-2-yl, benzothiazolyl, benzimidazolyl, fluorenyl, or thioxanthenyl. In this specification, an aryl group may have one or more optional substituents on its ring. Examples of the substituents include, but are not limited to, an alkoxy group, a halogen atom, an amino group, a mono- or di-substituted amino group, a substituted silyl group, or an acyl group. When an aryl group has two or more substituents, they may be the same or different. The same applies to the aryl moiety of other substituents (such as an aryloxy group or an arylalkyl group) that contain an aryl moiety.

[0016] In the present specification, the term "alkoxy group" refers to a structure in which the alkyl group is bonded to an oxygen atom, and examples thereof include saturated alkoxy groups that are linear, branched, cyclic, or a combination thereof. Suitable examples include methoxy, ethoxy, n-propoxy, isopropoxy, cyclopropoxy, n-butoxy, isobutoxy, s-butoxy, t-butoxy, cyclobutoxy, cyclopropylmethoxy, n-pentyloxy, cyclopentyloxy, cyclopropylethyloxy, cyclobutylmethyloxy, n-hexyloxy, cyclohexyloxy, cyclopropylpropyloxy, cyclobutylethyloxy, and cyclopentylmethyloxy.

[0017] In the present specification, "alkylene" refers to a divalent group consisting of a linear or branched saturated hydrocarbon, and examples thereof include methylene, 1-methylmethylene, 1,1-dimethylmethylene, ethylene, 1-methylethylene, 1-ethylethylene, 1,1-dimethylethylene, 1,2-dimethylethylene, 1,1-diethylethylene, 1,2-diethylethylene, 1-ethyl-2-methylethylene, trimethylene, 1-methyltrimethylene, 2-methyltrimethylene, 1,1-dimethyltrimethylene, 1,2 -dimethyltrimethylene, 2,2-dimethyltrimethylene, 1-ethyltrimethylene, 2-ethyltrimethylene, 1,1-diethyltrimethylene, 1,2-diethyltrimethylene, 2,2-diethyltrimethylene, 2-ethyl-2-methyltrimethylene, tetramethylene, 1-methyltetramethylene, 2-methyltetramethylene, 1,1-dimethyltetramethylene, 1,2-dimethyltetramethylene, 2,2-dimethyltetramethylene, 2,2-di-n-propyltrimethylene, and the like.

[0018] 1. A compound represented by general formula (I) or a salt thereof One embodiment of the present invention is a compound represented by the following general formula (I) or a salt thereof (hereinafter also referred to as "the compound of the present invention"). TIFF0007628340000003.tif44154

[0019] Without intending to be bound by theory, in the present invention, a cancer biomarker enzyme is targeted, its substrate site is incorporated into a drug molecule, and only when this is cleaved by an enzymatic reaction does a quinone methide intermediate become exposed, which is then tagged with an intracellular nucleophile, allowing the compound to remain in the cell. The molecular design of the compound was based on this idea. As a result, it was found that the compound represented by the above general formula (I) can selectively and sustainably maintain a high boron concentration in cancer cells, and is useful as a novel probe for BNCT. Figure 2 shows a schematic diagram of the mechanism by which the compound of the present invention remains in the cell after reacting with a cancer biomarker enzyme.

[0020] In general formula (I), Y is an enzyme recognition site, a part of which is cleaved by a cancer cell-specific enzyme activity to induce the formation of a quinone methide. Y can be selected depending on the type of the target enzyme. When the target enzyme, the cancer biomarker enzyme, is a glycosidase, Y is selected from a group derived from a saccharide, and when the target enzyme is a peptidase, Y is selected from a group derived from an amino acid or a group containing an amino acid.

[0021] In formula (I), Y is preferably -NH-CO-L, -NH-L' or -OL'. Here, L is a partial structure of an amino acid. The partial structure of an amino acid of L means that L, together with the C=O to which it is bonded, constitutes an amino acid, an amino acid residue, a peptide, or a part of an amino acid.

[0022] In the present specification, the term "amino acid" refers to any compound having both an amino group and a carboxyl group, including natural and non-natural amino acids. It may be any of neutral amino acids, basic amino acids, and acidic amino acids, and may be amino acids that function as transmitters such as neurotransmitters themselves, as well as amino acids that are components of polypeptide compounds such as physiologically active peptides (including dipeptides, tripeptides, tetrapeptides, and oligopeptides) and proteins, for example, α-amino acids, β-amino acids, and γ-amino acids. It is preferable to use optically active amino acids as the amino acids. For example, either D- or L-amino acids may be used for α-amino acids, but it may be preferable to select optically active amino acids that function in living organisms.

[0023] 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. The amino acid residue includes 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 and a dipeptide (a dipeptide consisting of an amino acid and proline) of a DPP4 substrate.

[0024] As used herein, the term "peptide" refers to a structure in which two or more amino acids are linked by peptide bonds. Preferred peptides include the above-mentioned DPP4 substrate dipeptides (dipeptides consisting of amino acid-proline; here, the amino acid is, for example, glycine, glutamic acid, or proline), etc.

[0025] An example of a case in which L, together with the C=O to which it is bonded, forms part of an amino acid is the above-mentioned γ-glutamyl group, in which the carboxyl group in the side chain of the amino acid is bonded to -NH2 to form a carbonyl group and becomes part of the amino acid.

[0026] L' is a saccharide or a partial structure of a saccharide, a saccharide having a self-cleaving linker, an amino acid or a peptide having a self-cleaving linker. The saccharide substructure of L', together with the O to which it is attached, constitutes a saccharide, a part of a saccharide.

[0027] Examples of sugars 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-DN-acetylglucosamine, β-DN-acetylgalactosamine, and the like, with β-D-galactose being preferred.

[0028] The self-cleavable linker means a linker that is spontaneously cleaved and decomposed, and examples thereof include carbamate, urea, para-aminobenzyloxy group, and ester group.

[0029] In one preferred aspect of the invention, Y has a structure selected from the following: 10. The compound or salt thereof according to claim 1, wherein Y has a structure selected from the following: TIFF0007628340000004.tif105167

[0030] In general formula (I), X acts as a leaving group that is eliminated from the benzene ring when a part of the enzyme recognition site of Y is cleaved by a cancer cell-specific enzyme activity, resulting in the formation of a quinone methide.

[0031] X is selected from the group consisting of a fluorine atom, an ester group (-OC(=O)-R'), a carbonate group (-OCO2-R'), a carbamate group (-OCONH-R'), phosphoric acid and its ester group (-OP(=O)(-OR')(-OR''), and sulfuric acid and its ester group (-OSO2-OR'). Here, R′ and R″ are each independently selected from a substituted or unsubstituted alkyl group, or a substituted or unsubstituted aryl group.

[0032] X is preferably a fluorine atom or an ester group (-OC(=O)-R'). Without intending to be bound by theory, when X is a fluorine atom or an ester group (-OC(=O)-R'), a quinone methide is rapidly formed when Y is cleaved.

[0033] R 1 and R 2 are each independently selected from a hydrogen atom or a monovalent substituent. The monovalent substituent is a halogen atom or an alkyl group having one or more carbon atoms (for example, an alkyl group having about 1 to 6 carbon atoms). R 1 and R 2 are preferably each independently selected from a hydrogen atom or a fluorine atom.

[0034] In the general formula (I), -Y is -C(R 1 )(R 2 It is preferable that -Y and -C(R 1 )(R 2 ) When X is in such a position on the benzene ring, a quinone methide structure can be formed when Y is cleaved.

[0035] R 3 represents a hydrogen atom or 1 to 3 identical or different monovalent substituents present on a benzene ring. R 3 The monovalent substituent is selected from the group consisting of an alkyl group having one or more carbon atoms (for example, an alkyl group having about 1 to 6 carbon atoms), an alkoxycarbonyl group (-C(=O)-OR'), a nitro group, an amino group, a hydroxyl group, an alkylamino group (-NHR', -NR'2), an alkoxy group (-OR'), an ester group (-O-CO-R'), an amide group (-NHCOR'), a halogen atom, a boryl group, and a cyano group. Here, R' is a substituted or unsubstituted alkyl group, or a substituted or unsubstituted aryl group. When there are two or more R', each R' may be the same or different.

[0036] In one aspect of the compounds of the invention, R 3 The monovalent substituent of is an alkyl group (e.g., a methyl group) or an alkoxy group (e.g., a methoxy group). It is preferable to introduce an electron-donating alkyl group or alkoxy group into the benzene ring, since it has excellent intracellular retention.

[0037] In one aspect of the compounds of the invention, R 3 The monovalent substituent of R is a halogen atom (preferably an iodine atom). 3 When is a halogen atom (preferably an iodine atom), it is possible to enhance the trapping effect on cells.

[0038] In one aspect of the compounds of the invention, R 3 one or more of the monovalent substituents of R is an alkyl group (e.g., a methyl group) or an alkoxy group (e.g., a methoxy group); 3 one or more of the monovalent substituents of is a halogen atom;

[0039] R 3 When R is the above-mentioned monovalent substituent, particularly an alkyl group or an alkoxy group, 3 The position of -C(R 1 )(R 2 ) The 5-position, which corresponds to the para-position of X, and / or the 4-position, which corresponds to the meta-position, are preferred.

[0040] In another aspect of the compounds of the invention, R 3 are all hydrogen atoms.

[0041] In the general formula (I), B is 10 It represents a group containing B. B is 10 Any group containing B may be used, and a boric acid residue ( 10 Although a group derived from a compound having one boron atom in the molecule, such as B(OH)2-, is acceptable, a group derived from a boron cluster is preferred.

[0042] The boron cluster may be any polyhedral structure that can be used in boron neutron capture therapy. For example, closododecaborate ([B 12 H 12 ] 2- ), ionic closocarborane ([CB 11 H 12 ] - ), fat-soluble closocarborane ([C2B 10 H 12 ]), nidocarborane ([C2B9H 11 ] - ), bisdicarbolide metal complexes ([(C2B9H 11 )2M] (M is metal), GB10 ([B 10 H 12 ] 2- ), 1,2-dicarbacloso-dodecaborane, 1,7-dicarbacloso-dodecaborane, 1,12-dicarbacloso-dodecaborane, dicarbacloso-decarborane ([C2B8H 10 ]), sulfur-substituted undecahydrododecaborate, and the like, but are not limited thereto.

[0043] All boron atoms in a boron cluster are 10 B may be used, but only a part of it may be used. 10 It may also be B. In this specification, the expression "a group derived from a boron cluster" is used, which means, for example, a group derived by removing one hydrogen atom in a boron cluster.

[0044] In formula (I), Z represents a single bond or a linking group. Here, when Z is a "single bond", this means that B is directly bonded to the benzene ring without a linking group.

[0045] The linking group may be any group that functions as a linker and is metabolically stable, but is preferably selected from the group consisting of an alkylene group (wherein one or more -CH2- of the alkylene group may be substituted with -O-, -S-, -NH-, or -CO-), an arylene (including heteroarylene), a cycloalkylene (e.g., cyclohexylene), an alkoxyl group, a polyethylene glycol chain, and a group formed by arbitrarily bonding two or more groups selected from these groups. The number of carbon atoms in the alkylene group is not particularly limited, but is preferably 5 to 20, and more preferably 5 to 15. When -CH2- in the alkylene group is replaced with -O-, -S-, -NH-, or -CO-, these groups are counted as having one carbon atom and are included in the above-mentioned "number of carbon atoms in the alkylene group." In addition, arylene includes those that use a benzene ring as a linker, such as a phenylene group, and divalent linkers derived from aromatic and cyclic hydrocarbons containing heterocycles.

[0046] In one preferred embodiment of the compound of the present invention, the linking group is an alkylene group (with the proviso that one or more -CH2- of the alkylene group may be replaced with -O-, -S-, -NH-, or -CO-).

[0047] The position at which BZ- is introduced is not particularly limited, but it is preferable that BZ- is bonded to the meta or para position of the benzene ring relative to Y, because BZ- is metabolically stable and may not serve as a substrate for the target enzyme if it is too close to the enzyme recognition site.

[0048] Non-limiting examples of the compounds of the present invention are shown below, but the compounds of the present invention are not limited thereto. TIFF0007628340000005.tif145166 is a group derived from closocarborane (o-carborane) (where the grey atoms are BH and the black atoms are C).

[0049] Unless otherwise specified, the compound represented by general formula (I) also includes its 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 carbons, the stereochemistry of the asymmetric carbons can be either the (R) or (S) form, and the derivative may exist as a stereoisomer such as an enantiomer or diastereoisomer. Therefore, as the active ingredient of the probe for BNCT of the present invention, any stereoisomer in a pure form, any mixture of stereoisomers, racemate, etc. can be used, and all of them are included in the scope of the present invention.

[0050] The method for producing the compound represented by the general formula (I) is not particularly limited, but the synthesis method for the representative compounds among the compounds included in the general formula (I) is specifically shown in the examples of this specification. Those skilled in the art can produce the compounds included in the formula (I) by appropriately modifying or altering the starting materials, reaction reagents, reaction conditions, etc. as necessary while referring to the examples of this specification and the following schemes.

[0051] 2. Pharmaceutical Compositions Another embodiment of the present invention is a pharmaceutical composition comprising a compound of the present invention or a pharma- ceutically acceptable salt thereof (hereinafter also referred to as "the pharmaceutical composition of the present invention"). A preferred embodiment of the pharmaceutical composition of the present invention is a pharmaceutical composition used for boron neutron capture therapy.

[0052] The pharmaceutical composition of the present invention is preferably a pharmaceutical composition used in boron neutron capture therapy, which can accumulate in cancer cells by acting selectively on cancer cells through cancer cell-specific enzymatic activity.

[0053] The cancer cell-specific enzyme is a peptidase or a glycosidase. Examples of peptidases include γ-glutamyl transpeptidase (GGT), dipeptidyl peptidase IV (DPP-IV), and calpain. Examples of glycosidases include β-galactosidase, β-glucosidase, α-mannosidase, α-L-fucosidase, β-hexosaminidase, β-N-acetylgalactosaminidase, and the like.

[0054] The pharmaceutical composition of the present invention may contain not only the compound represented by the general formula (I) but also its salt or a solvate or hydrate thereof. The salt is not particularly limited as long as it is a pharma- ceutically acceptable salt, and may include, for example, a base addition salt, an acid addition salt, an amino acid salt, etc. Examples of the base addition salt include, for example, alkaline earth metal salts such as sodium salt, potassium salt, calcium salt, magnesium salt, etc., ammonium salt, or organic amine salts such as triethylamine salt, piperidine salt, morpholine salt, etc. Examples of the acid addition salt include, for example, mineral acid salts such as hydrochloride, hydrobromide, sulfate salt, nitrate salt, phosphate salt, etc.; 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, salicylic acid, etc. Examples of the amino acid salt include glycine salt, aspartate salt, glutamate salt, etc. Metal salts such as aluminum salts may also be used.

[0055] The type of solvent that forms the solvate is not particularly limited, but examples include ethanol, acetone, and isopropanol.

[0056] The pharmaceutical composition of the present invention is used for boron neutron capture therapy. That is, the pharmaceutical composition of the present invention is administered to humans or non-human animals (mouse, rat, hamster, rabbit, cat, dog, cow, sheep, monkey, etc.), and then irradiated with low-energy thermal neutrons, thereby selectively destroying tumor cells. The diseases to be treated include, but are not limited to, malignant tumors, such as brain tumors, malignant melanoma, head and neck cancer, lung cancer, liver cancer, thyroid cancer, skin cancer, bladder cancer, mesothelioma, pancreatic cancer, breast cancer, meningioma, sarcoma, etc.

[0057] When used as a pharmaceutical composition containing the compound represented by general formula (I) or a pharma- ceutical acceptable salt thereof, it can be formulated by mixing with a pharma- ceutical acceptable carrier or diluent according to a known method. The dosage form is not particularly limited, and can be an injection, tablet, powder, granule, capsule, liquid, suppository, sustained release agent, etc. The administration method is also not particularly limited, and can be administered orally or parenterally (administration by injection or drip into intradermal, intraperitoneal, vein, artery, or spinal fluid, etc.). These preparations are prepared according to conventional methods. Liquid preparations may be dissolved or suspended in water or other suitable solvents when used. Tablets and granules may 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 necessary, and buffers and preservatives may be added.

[0058] The dosage of the pharmaceutical composition of the present invention varies depending on the subject of administration, the administration method, etc., but for example, when administered to an adult as an injection, the compound can be administered in one or several divided doses for a single treatment so that the compound is administered in a single dose of 10 to 1,000 mg / kg.

[0059] Another embodiment of the invention is a method for diagnosing, treating, or diagnosing and treating a disease or a condition that may lead to a disease, comprising: (A) administering to a subject having or suspected of having a disease or condition a pharmaceutical composition of the invention; and (B) localized to a target tissue of the subject. 10 Irradiating the B atoms with neutrons, thereby performing boron neutron capture therapy on the target tissue. (hereinafter also referred to as "the method of the present invention")

[0060] In the method of the present invention, the dosage of the pharmaceutical composition of the present invention is as described above.

[0061] In the method of the present invention, 10 To irradiate B atoms with neutrons, a nuclear reactor or accelerator-type neutron generator commonly used in BNCT is used, and the conditions required for treatment, such as the neutron dose, neutron spectrum, and exposure time, are determined. The energy of the irradiated neutrons is usually around 0.025 eV for thermal neutrons and 0.5 eV to 40 keV for epithermal neutrons. EXAMPLES

[0062] The present invention will be described below with reference to examples, but the present invention is not limited thereto.

[0063] General Procedure and Materials All reagents and dry solvents were purchased from commercial suppliers (Tokyo Chemical Industry Co., Ltd., Fujifilm Wako Pure Chemical Industries, Ltd., Sigma-Aldrich, Kanto Chemical Co., Ltd., Dojindo Laboratories, Inc., Watanabe Chemical Industries, Ltd., Gibco, Invitrogen, Thermo scientific, Merck) and used without further purification.

[0064] Instruments used The progress of the reaction was monitored by TLC silica gel 60F254 (Merck Inc.) and ACQUITY UPLC / MS system (Waters Inc.). 1 HNMR and 13 CNMR spectra were obtained using a JEOL JNM-ECZ400 ( 1 400MHz for H NMR; 13 CNMR was measured at 100 MHz; σ values ​​are in ppm relative to tetramethylsilane (TMS). Mass spectra (MS) were measured on a JEOL JMS-T100 LC AccuToF (ESI). Column chromatography using silica gel was performed using an MPLC system (Yamazen Smart Flash EPCLC W-Prep 2XY, Tokyo, Japan). Reverse-phase MPLC purification was performed on an Isolera™ One (Biotage) equipped with a SNAP Ultra C18 (Biotage). Preparative HPLC was performed on an Inertsil ODS-3 (10.0 × 250 mm) column (GL Sciences Inc.) using a HPLC system consisting of a pump (PU-2086, JASCO) and a detector (MD-2015 or FP-2025, JASCO) with eluent A (HO containing 0.1% TFA (v / v)) and eluent B (20% HO containing 0.1% TFA (v / v) in CH3CN) or eluent C (HO containing 100 mM TEAA, i.e., 100 mM trimethylamine and aqueous acetic acid) and eluent D (20% HO containing 100 mM TEAA in CH3CN) at a flow rate of 3 or 5 mL / min.

[0065] Enzyme assays (LC / MS analysis) In screw-cap vials for LC / MS analysis (Agilent Technologies Inc.), phosphate buffered saline (pH 7.4) or 0.1 M HEPES buffer (pH 7.4) containing prodrug (100 μM) in the presence or absence of inhibitors (GGsTop® at 100 μM for GGT and 200 μM for DPP-IV) and DMSO (1%) as a cosolvent were prepared. Enzymes (1 U / mL for GGT and >0.1 mU / mL for DPP-IV) were added and then incubated at 37° C. for 12 hours. LC / MS analysis (SIM) was performed under the following conditions (eluent A: HO containing 0.01 M ammonium formate, eluent B: 80% acetonitrile / HO containing 0.01 M ammonium formate, A / B=90 / 10→0 / 100 in 12 minutes). In experiments evaluating the reaction of azaquinone methides with nucleophiles (GSH, l-Cys), 0.1 M HEPES buffer (pH 7.4) containing 5 mM of each nucleophile was prepared and used.

[0066] cell culture H226, H460, SHIN3 and SKOV3 cells were cultured in RPMI 1640 medium (Roswell Park Memorial Institute 1640 medium, Gibco) containing 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin (Gibco). A549, Hela and HepG2 cells were cultured in DMEM (Dulbecco's Modified Eagle Medium, Gibco) containing 10% fetal bovine serum and 1% penicillin-streptomycin. Caco-2 cells were cultured in DMEM containing 20% ​​fetal bovine serum, 1% penicillin-streptomycin and 1% MEM non-essential amino acid solution (100X). All cells were cultured in a 37°C, 5% CO2 incubator.

[0067] Cell viability test (CCK-8 assay) cells (1.0×10 4 Cells / well) were grown in 96-well plates for 24 h and treated with prodrugs across a range of concentrations (0 [control] or 1, 2.5, 5, 10, 25, 50). After 24 h, the extent of cell proliferation was assessed using a CCK-8 assay (Dojindo Laboratories, Tokyo, Japan). CCK-8 solution (10 μL) was added to each well, followed by incubation at 37°C in 5% CO2 for 2 h. Absorbance at 440 nm was measured by an Envision2103 multilabel reader (Prekin Elmer). Cell viability was expressed as a percentage of control cells. For each concentration of prodrug, the average of the mean absorbance from triplicate wells was calculated.

[0068] Cellular uptake To assess cellular uptake, H226 cells were plated in 6-well plates (2.5 × 10 5 cells / mL, 5.0×10 5Cells were seeded on 100 μL of EP-4OCB-FMA (10 μM) and incubated for 24 h. After removing the medium, the cells were incubated for 3 h in the presence or absence of sitagliptin (100 μM) in medium containing EP-4OCB-FMA (10 μM) and 1% DMSO. Then, 100 μL of the supernatant was taken and diluted with 900 μL of 5.5% nitric acid to be used as sample "supernatant". After removing the remaining supernatant, the cells were washed with PBS and detached by incubating in 0.2 ml of 0.05% trypsin / EDTA solution. The cell suspension was mixed with 2 mL of medium and the cells were collected by centrifugation. After carefully removing the supernatant, 2 mL of medium was added and the cell number was counted. Then, the cells were dissolved in 400 μL of 60% nitric acid and the lysate was heated to 90 °C for ashing. The ashed sample was diluted with 4.4 mL of ultrapure water to be used as sample "cell". The amount of boron in the samples was quantified using MP-AES (Agilent 4100 MP-AES, Agilent Technologies Inc., Santa Clara, CA, USA). In experiments to evaluate intracellular boron retention, cells were first incubated with EP-4OCB-FMA without sitagliptin as described above for 3 h. Then, cells were incubated in fresh medium without EP-4OCB-FMA for 30 min to release intracellular boron, followed by harvesting with 0.05% trypsin / EDTA. The subsequent procedures were the same as the measurement method described above.

[0069] [Synthesis Example 1] The compound of the present invention, gGlu-4OCB-FMA (Compound 18), was synthesized as a GGT candidate drug according to the following synthesis scheme 1. Details of each reaction step are described below.

[0070] Synthetic Scheme 1 TIFF0007628340000006.tif117170

[0071] (1) Synthesis of compound 10 o-Carborane (534 mg, 3.70 mmol) was dissolved in 12 mL of anhydrous THF. After cooling to -78 °C, a solution of 1.6 Mn-BuLi in THF (2.5 mL, 4.0 mmol) was added dropwise to the solution. After stirring for 30 min at -78 °C under argon atmosphere, a solution of 1.2 M ethylene oxide in THF (5.0 mL, 6.0 mmol) was added dropwise over 10 min, and the reaction solution was then warmed to 0 °C. After stirring for 1 h, 4 mL of saturated aqueous NH4Cl was added. After stirring for several tens of min, the reaction solution was extracted three times with AcOEt. The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by MPLC (OH silica gel, AcOEt / hexane) to give a pale yellow oil (375.3 mg, 1.98 mmol, 54%). 1 H-NMR (400 MHz, CDCl3) δ 3.98 (s, 1H), 3.79 (t, J = 5.9 Hz, 2H), 2.49 (t, J = 5.9 Hz, 2H),1.68 (s, 1H), 1.2-3.2 (br, 10H) 13 C-NMR (101 MHz, CDCl3) δ73.10, 60.74, 60.49, 39.85

[0072] (2) Synthesis of compound 11 5-Bromo-2-nitrobenzaldehyde (1.00 g, 4.35 mmol) was dissolved in 20 mL of dry MeOH, then sodium borohydride (163 mg, 4.30 mmol) was added in portions at 0° C. The solution was stirred at room temperature for 30 min. After warming to room temperature, stirring was continued for 10 min. The reaction was quenched with water, and the solvent was then evaporated. The mixture was extracted with AcOEt, then the organic layer was washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by MPLC (OH silica gel, AcOEt / Hexane) to give a white solid (1211 mg, 5.22 mmol, quantitative yield).

[0073] (3) Synthesis of compound 12 Compound 11 (602 mg, 2.59 mmol), TBSCl (781 mg, 5.18 mmol) and imidazole (531 mg, 7.80 mmol) were added to 25 mL of dry toluene and 5 mL of dry CHCl. ​​The solution was stirred at room temperature under argon for 16 h. AcOEt was added to the solvent and the organic layer was washed twice with water. The organic layer was dried over NaSO and concentrated under reduced pressure. The residue was purified by MPLC (OH silica gel, AcOEt / hexane) to give an orange oil (838 mg, 2.42 mmol, 93%).

[0074] (4) Synthesis of compound 13 Compound 12 (401 mg, 1.16 mmol) and compound 10 (283 mg, 1.50 mmol) were dissolved in 2 mL of dry toluene, then Cs2CO3 (549 mg, 1.75 mmol) and 2-di-t-butylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl (6.4 mg, 0.015 mmol) were added. After degassing by freeze-pump-thaw cycles, Pd2(dba)3 (6.9 mg, 0.0075 mmol) was added to the solution. The reaction solution was stirred at 85 °C under argon atmosphere for 2 h. AcOEt was added to the solvent, and the organic layer was washed with water. The organic layer was dried over Na2SO4 and concentrated under low pressure. The residue was purified by MPLC (OH silica gel, AcOEt / hexane) to give an orange solid (384 mg, 0.845 mmol, 73%). 1 H-NMR (400 MHz, CDCl3) δ 8.18 (d, J = 9.0 Hz, 1H), 7.42 (d, J = 3.2 Hz, 1H), 6.82 (dd, J = 9.0, 3.2 Hz, 1H), 5.11 (s, 2H), 4.17 (t, J = 6.2 Hz, 2H), 3.79 (s, 1H), 2.79 (t, J = 6.2 Hz, 2H), 1.40-3.20 (br, 10H), 1.00 (s, 9H), 0.16 (s, 6H). 13C-NMR (101 MHz, CDCl3) δ 162.1, 142.5, 140.1, 127.8, 113.1, 112.7, 72.1, 66.1, 62.4, 60.8, 37.1, 26.1, 18.5, -5.3; HRMS Calcd for 12 C 17 1 H 34 10 B2 11 B8 14 N 16 O4 28 Si: 452.32603 [MH] - ; Found: 452.32608 (+0.05 mDa).

[0075] (5) Synthesis of compound 14 Compound 13 (384 mg, 0.846 mmol) was dissolved in 13 mL of ethanol and 12 mL of water, then ammonium chloride (144 mg, 2.68 mmol) and Fe (195 mg, 3.49 mmol) were added. After stirring at room temperature for 1 h, the solvent was stirred at 75 °C for 3 h. Ethanol was removed by evaporation. The remaining solution was extracted with AcOEt and the organic layer was washed with saturated aqueous NaHCO3. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by MPLC (OH silica gel, AcOEt / hexane) to give an orange solid (196 mg, 0.463 mmol, 55%). 1 H-NMR (400 MHz, CDCl3) δ 6.61-6.64 (m, 3H), 4.63 (s, 2H), 3.98 (t, J = 5.7 Hz, 2H), 3.90 (s, 1H), 2.68 (t, J = 5.7 Hz, 2H), 1.40-3.20 (br, 10H), 0.91 (s, 9H), 0.09 (s, 6H). 13 C-NMR (101 MHz, CDCl3) δ 150.2, 140.4, 127.0, 116.9, 115.2, 114.6, 73.0, 66.4, 64.4, 60.3, 37.41, 26.0, 18.4, -5.4

[0076] (6) Synthesis of compound 15 Compound 14 (177 mg, 0.418 mmol) and N-Alloc-Glu(OH)-OAllyl (141 mg, 0.520 mmol) were dissolved in 6 mL of dry THF, followed by the addition of HATU (490 mg, 1.29 mmol) and DIEA (225 μL, 1.29 mmol). The solution was stirred at room temperature under argon for 8 h. The solvent was removed by evaporation. AcOEt was added to the residue and the organic layer was washed with water. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by MPLC (OH silica gel, AcOEt / Hexane) to give a white solid (276 mg, 0.408 mmol, 98%). 1 H-NMR (400 MHz, CDCl3) δ 8.64 (s, 1H), 7.99 (d, J = 8.8 Hz, 1H), 6.76 (dd, J = 8.8, 3.0 Hz, 1H), 6.66 (d, J = 3.0 Hz, 1H), 5.84-5.94 (m, 2H), 5.63 (d, J = 7.9 Hz, 1H), 5.18-5.35 (m, 4H), 4.68 (s, 2H), 4.65 (d, J = 5.0 Hz, 2H), 4.56 (d, J = 4.6 Hz, 2H), 4.43 (m, 1H), 4.04 (t, J = 5.7 Hz, 2H), 3.84 (s, 1H), 2.71 (t, J = 5.7 Hz, 2H), 2.31-2.53 (m, 3H), 2.07-2.15 (m, 1H), 0.92 (s, 9H), 0.10 (s, 6H)

[0077] (7) Synthesis of compound 16 Compound 15 (243 mg, 0.359 mmol) was dissolved in 20 mL of dry THF, followed by the addition of TBAF (~1 M in THF) (914 μL, 0.914 mmol) and acetic acid (34.9 μL, 0.610 mmol). The solution was stirred at room temperature under argon for 45 min and the solvent was removed by evaporation. AcOEt was added to the residue and the organic layer was washed with water. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by MPLC (OH silica gel, AcOEt / hexane) to give a white solid (132 mg, 0.235 mmol, 65%). 1 H-NMR (400 MHz, CDCl3) δ 8.49 (s, 1H), 7.73 (d, J = 8.2 Hz, 1H), 6.77-6.80 (m, 2H), 5.83-5.95 (m, 2H), 5.70 (brs, 1H), 5.20-5.36 (m, 4H), 4.54-4.65 (m, 6H), 4.42 (m, 1H), 4.04 (t, J = 5.7 Hz, 2H), 3.85 (s, 1H), 3.22 (brs, 1H), 2.71 (t, J = 5.7 Hz, 2H), 2.33-2.50 (m, 3H), 1.98-2.07 (m, 1H), 1.40-3.20 (br, 10H). 13 C-NMR (101 MHz, CDCl3) δ 171.68, 171.03, 156.57, 154.78, 132.40, 131.37, 130.47, 125.33, 119.36, 118.20, 115.28, 114.10, 72.68, 66.42, 66.21, 65.79, 63.59, 60.41, 53.58, 37.21, 33.46, 29.00.

[0078] (8) Synthesis of compound 17 Compound 16 (48 mg, 0.085 mmol) was dissolved in 3 mL of dry CHCl, then 2 mL of Deoxofluor® (31.6 μL, 0.171 mmol) in dry CHCl was added at 0° C. The solution was stirred at room temperature under argon for 30 min, and CHCl was added to the reaction solution and washed with saturated aqueous NaHCO. The organic layer was dried over NaSO and concentrated under reduced pressure. The residue was purified by MPLC (OH silica gel, AcOEt / hexane) to give a white solid (34 mg, 0.060 mmol, 71%). 1 H-NMR (400 MHz, CDCl3) δ 7.80 (s, 1H), 7.63 (d, J = 9.1 Hz, 1H), 6.85 (m, 2H), 5.85-5.95 (m, 2H), 5.63 (d, J = 7.3 Hz, 1H), 5.20-5.47 (m, 6H), 4.65 (d, J = 5.9 Hz, 2H), 4.57 (d, J = 5.5 Hz, 2H), 4.44-4.47 (m, 1H), 4.06 (t, J = 5.7 Hz, 2H), 3.83 (s, 1H), 2.72 (t, J = 5.7 Hz, 2H), 2.50 (m, 2H), 2.31-2.38 (m, 1H), 1.96-2.07 (m, 1H), 1.40-3.20 (br, 10H). 13 C-NMR (101 MHz, CDCl3) δ 171.64, 170.83, 156.42, 155.17, 132.49, 131.40, 130.57, 130.43, 129.64, 126.46, 119.31, 118.13, 115.28, 114.84, 114.77, 83.44, 81.80, 72.59, 66.37, 66.15, 65.88, 60.42, 53.50, 37.22, 33.11, 29.00.

[0079] (9) Synthesis of compound 18 Compound 17 (17 mg, 0.030 mmol) was dissolved in 5 mL of dry CHCl, then 1,3-dimethylbarbituric acid (46 mg, 0.30 mmol) and tetrakis (9.7 mg, 0.084 mmol) were added. The solution was stirred at room temperature under argon for 4 h. The reaction solution was concentrated by evaporation. The residue was purified by HPLC (eluent: A: H0, 0.1% TFA (v / v), eluent B: CHCN / H0 = 80 / 20, 0.1% TFA (v / v)) to give a white solid (8.6 mg, 0.020 mmol, 65%). 1 H-NMR (400 MHz, MeOH-D4) δ 7.25 (d, J = 8.7 Hz, 1H), 7.03 (d, J = 2.7 Hz, 1H), 6.94 (dd, J = 8.7, 2.7 Hz, 1H), 5.34 (d, J = 47.6 Hz, 2H), 4.59 (s, 1H), 4.11 (t, J = 5.8 Hz, 2H), 4.03 (t, J = 6.4 Hz, 1H), 2.78 (t, J = 5.8 Hz, 2H), 2.70 (t, J = 7.3 Hz, 2H), 2.19-2.30 (m, 2H). 13 C-NMR (101 MHz, MeOH-D4) δ 172.32, 170.35, 156.73, 133.89, 133.72, 127.83, 114.65, 114.63, 113.74, 113.66, 81.75, 80.12, 73.36, 65.67, 61.94, 52.26, 36.77, 31.05, 25.78.

[0080] [Example 1] Confirmation of reactivity of gGlu-4OCB-FMA with purified enzyme To verify whether the gGlu-4OCB-FMA synthesized above can be a substrate for GGT, an enzymatic reaction was carried out with purified enzymes, and the products were analyzed by LC / MS. As a result, gGlu-4OCB-FMA was completely consumed by the addition of GGT, and a benzyl alcohol was confirmed as the product (Figure 3). This is thought to be due to the reaction of the generated azaquinone methide intermediate with water molecules in the buffer as a nucleophile, and is a result indirectly indicating the generation of the azaquinone methide intermediate. In addition, since the enzyme reaction did not proceed in the presence of GGsTop (registered trademark), a GGT inhibitor, it was confirmed that gGlu-4OCB-FMA serves as a substrate for GGT. FIG. 3 shows LC / MS analysis of the enzymatic reaction of gGlu-4OCB-FMA (100 μM): mass chromatograms of gGlu-4OCB-FMA and product compounds after incubation for 12 h at 37° C. in PBS (pH 7.4) (1% DMSO as a co-solvent) in the presence or absence of GGT (1 U / mL) and GGsTop® (100 μM).

[0081] [Synthesis Example 2] Next, the target enzyme of the candidate compound was changed to dipeptidyl peptidase IV (DPP-IV), an aminopeptidase whose activity has been reported to be enhanced in various cancer cells, and based on the same drug design, the compound of the present invention, EP-4OCB-FMA (compound 25), was synthesized according to the following synthesis scheme 2. Here, DPP-IV is known to recognize various two amino acid residues as substrates, but the Glu-Pro sequence is considered to be a highly water-soluble structure, and therefore this sequence was adopted as the substrate site.

[0082] Synthetic Scheme 2 TIFF0007628340000007.tif92168

[0083] (1) Synthesis of compound 22 Compound 14 (125.4 mg, 0.2960 mmol) and Boc-E(OtBu)-P-OH (142.4 mg, 0.3556 mmol) were dissolved in 2 mL of dry THF, followed by the addition of HATU (341.2 mg, 0.8973 mmol) and DIEA (155 μL, 0.888 mmol). The solution was stirred at room temperature under argon for 3 h. The solvent was removed by evaporation. AcOEt was added to the residue and the organic layer was washed with brine. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by MPLC (OH silica gel, AcOEt / hexane) to give a white solid (227.8 mg, 0.283 mmol, 96%). 1 H-NMR (400 MHz, CDCl3) δ 8.82 (s, 1H), 7.81 (d, J = 8.7 Hz, 1H), 6.80 (d, J = 2.7 Hz, 1H), 6.72 (dd, J = 8.7, 2.7 Hz, 1H), 5.23 (d, J = 9.1 Hz, 1H), 4.75 (d, HBn, J gem = 13.3 Hz, 1H), 4.51-4.64 (m, 3H), 4.03 (t, J = 5.7 Hz, 2H), 3.85 (s, 1H), 3.75-3.78 (m, 2H), 2.71 (t, J = 5.7 Hz, 2H), 2.27-2.42 (m, 3H), 2.15-2.24 (m, 1H), 1.98-2.11 (m, 3H), 1.69-1.79 (m, 1H), 1.44 (s, 9H), 1.43(s, 9H), 1.40-3.20 (br, 10H), 0.93 (s, 9H), 0.13 (s, 3H), 0.09 (s, 3H). 13C-NMR (101 MHz, CDCl3) δ 172.20, 172.09, 169.37, 155.67, 154.33, 133.45, 130.01, 124.25, 113.72, 113.14, 80.69, 79.82, 72.74, 65.75, 63.44, 60.88, 60.28, 51.22, 47.48, 37.19, 30.98, 28.41, 28.30, 28.16, 28.03, 25.94, 25.25, 18.36, -5.12; LRMS 806.58[C 36 H 67 10 B2 11 B8N3O8Si+H] +

[0084] (2) Synthesis of compound 23 Compound 22 (203 mg, 0.252 mmol) was dissolved in 15 mL of dry THF, followed by the addition of TBAF (~1 M in THF) (757 μL, 0.757 mmol) and AcOH (28.9 μL, 0.505 mmol). The solution was stirred at room temperature under argon for 45 min and the solvent was removed by evaporation. AcOEt was added to the residue and the organic layer was washed with brine. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by MPLC (OH silica gel, AcOEt / hexane) to give a white solid (109.6 mg, 0.158 mmol, 63%). 1 H-NMR (400 MHz, CDCl3) δ 9.10 (s, 1H), 7.78 (d, J = 8.2 Hz, 1H), 6.74-6.78 (m, 2H), 5.29 (d, J = 8.2 Hz, 1H), 4.73 (dd, J = 7.8, 2.7 Hz, 1H), 4.55-4.58 (m, 3H), 4.03 (t, J = 5.5 Hz, 2H), 3.73-3.84 (m, 4H), 2.71 (t, J = 5.5 Hz, 2H), 2.05-2.41 (m, 5H), 1.78-1.85 (m, 1H), 1.44 (s, 9H), 1.41 (s, 9H), 1.40-3.20 (br, 10H). 13C-NMR (101 MHz, CDCl3) δ 173.16, 172.66, 169.73, 155.55, 154.56, 133.28, 130.50, 124.81, 115.15, 113.91, 81.37, 80.08, 72.72, 65.75, 63.55, 61.24, 60.28, 51.22, 47.65, 37.22, 30.91, 28.41, 28.30, 28.08, 27.89, 25.17

[0085] (3) Synthesis of compound 24 Compound 23 (96.5 mg, 0.139 mmol) was dissolved in 6 mL of dry CHCl, then Deoxofluor® (51.4 μL, 0.279 mmol) was added at 0° C. The solution was stirred at room temperature under argon for 1 h. CHCl was added to the reaction solution and washed with saturated aqueous NaHCO. The organic layer was dried over NaSO and concentrated under reduced pressure. The residue was purified by MPLC (OH silica gel, AcOEt / hexane) to give a clear solid (79.8 mg, 0.115 mmol, 83%). 1 H-NMR (400 MHz, CDCl3) δ 8.81 (s, 1H), 7.67 (d, J = 8.2 Hz, 1H), 6.84 (m, 2H), 5.27-5.39 (m, 3H), 4.76 (dd, J = 7.8, 2.3 Hz, 1H), 4.55 (td, J = 9.0, 3.8 Hz, 1H), 4.05 (t, J = 5.7 Hz, 2H), 3.84 (s, 1H), 3.74 (m, 2H), 1.40-3.20 (br, 10H), 2.72 (t, J = 5.7 Hz, 2H), 2.46-2.51 (m, 1H), 2.27-2.43 (m, 2H), 1.95-2.20 (m, 4H), 1.75-1.81 (m, 1H), 1.44 (s, 9H), 1.42 (s, 9H). 13C-NMR (101 MHz, CDCl3) δ 173.22, 172.20, 169.53, 155.63, 154.93, 130.00, 129.62, 125.78, 115.22, 114.80, 114.72, 82.85, LRMS 806.58[C 30 H 52 10 B2 11 B8FN3O7+H] +

[0086] (4) Synthesis of compound 25 Compound 24 (50.8 mg, 0.0732 mmol) was dissolved in 1 mL of TFA. The solution was stirred at room temperature for 10 min. The reaction solution was diluted with CHCl and concentrated by evaporation. The residue was purified by HPLC (eluent: A: H0, 0.1% TFA (v / v), eluent B: CHCN / H0=80 / 20, 0.1% TFA (v / v)) to give a white solid (34.3 mg, 0.0638 mmol, 87%). 1 H-NMR (400 MHz, MeOH-D4) δ 7.22 (d, J = 8.7 Hz, 1H), 7.03 (d, J = 2.7 Hz, 1H), 6.93 (dd, J = 8.7, 2.3 Hz, 1H), 5.24-5.48 (m, 2H), 4.58-4.63 (m, 2H), 4.38 (dd, J = 7.3, 5.0 Hz, 1H), 4.10 (t, J = 5.7 Hz, 2H), 3.69-3.78 (m, 2H), 1.40-3.20 (br, 10H), 2.77 (t, J = 5.7 Hz, 2H), 2.59 (t, J = 7.3 Hz, 2H), 2.36-2.42 (m, 1H), 2.00-2.24 (m, 5H). 13C-NMR (101 MHz, Acetonitrile-d3) δ 175.16, 170.76, 167.49, 156.20, 133.20, 133.04, 128.19, 128.14, 127.32, 115.00, 114.23, 114.15, 82.27, 80.65, 73.83, 65.80, 62.04, 60.93, 51.51, 47.49, 36.66, 29.11, 28.98, 24.98, 24.84; HRMS Calcd for 12 C 21 1 H 37 10 B2 11 B8 19 F 14 N3 16 O5: 538.37204 [M+H] + ; Found: 538.37055 (-1.49 mDa).

[0087] [Reference synthesis example 1] A control compound, EP-4OCB-MA (compound 26), was synthesized according to the following synthesis scheme 3. Details of each reaction step are described below.

[0088] Synthetic Scheme 3 TIFF0007628340000008.tif26159

[0089] (1) Synthesis of compound 26 Compound 20 (62 mg, 0.21 mmol) and N-Boc-E(OtBu)-P-OH (124 mg, 0.310 mmol) were dissolved in a few mL of dry THF, followed by the addition of HATU (241 mg, 0.634 mmol) and DIEA (110.5 μL, 0.633 mmol). The solution was stirred at room temperature under argon for 3 h. The solvent was removed by evaporation. AcOEt and water were added to the residue and the organic layer was extracted. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The crude compound was dissolved in 2 mL of TFA. The solution was stirred at room temperature for 10 min and the reaction solution was concentrated by evaporation. The residue was purified by HPLC (eluent: A: H2O, 0.1% TFA (v / v), eluent B: CH3CN / H2O = 80 / 20, 0.1% TFA (v / v)) to give a white solid (84 mg, 0.16 mmol, 76%, 2 steps). 1 H-NMR (400 MHz, MeOH-D4) δ 7.16 (d, J = 8.6 Hz, 1H), 6.80 (d, J = 2.7 Hz, 1H), 6.73 (dd, J = 8.6, 2.7 Hz, 1H), 4.65 (dd, J = 8.2, 5.5 Hz, 1H), 4.58 (s, 1H), 4.38 (dd, J = 7.3, 5.0 Hz, 1H), 4.05 (t, J = 5.9 Hz, 2H), 3.68-3.79 (m, 2H), 2.75 (t, J = 5.7 Hz, 2H), 2.59 (t, J = 7.1 Hz, 2H), 2.36-2.41 (m, 1H), 2.02-2.26 (m, 8H), 1.40-3.20 (br, 10H). 13 C-NMR (101 MHz, MeOH-D4) δ 174.66, 171.77, 167.22, 156.59, 135.64, 128.52, 127.35, 115.93, 111.69, 73.46, 65.44, 61.86, 60.58, 50.92, 48.31, 48.10, 47.88, 47.67, 47.46, 47.25, 47.03, 36.80, 29.60, 28.12, 25.28, 24.91, 17.05

[0090] [Example 2] Confirmation of reactivity of EP-4OCB-FMA with purified enzyme To evaluate whether the EP-4OCB-FMA and EP-4OCB-MA synthesized above could serve as substrates for DPP-IV, we reacted them with the purified enzyme and then analyzed the products by LC / MS. First, the results for EP-4OCB-FMA are shown below. EP-4OCB-FMA was completely consumed by the addition of DPP-IV, and the benzyl alcohol was confirmed as the product (Figure 4). This is thought to be due to the reaction of the generated azaquinone methide intermediate with water molecules in the buffer as a nucleophile, and is a result indirectly indicating the generation of the azaquinone methide intermediate. In addition, the enzyme reaction was suppressed in the presence of sitagliptin, a DPP-IV inhibitor, confirming that EP-4OCB-FMA serves as a substrate for DPP-IV. Here, FIG. 4 shows LC / MS analysis of the enzymatic reaction of EP-4OCB-FMA (100 μM): mass chromatograms of EP-4OCB-FMA and product compounds after incubation in PBS (pH 7.4) (1% DMSO as a co-solvent) for 12 hours at 37° C. in the presence or absence of DPP-IV (>0.1 U / mL) and in the presence or absence of sitagliptin (200 μM).

[0091] Similarly, it was confirmed that the addition of DPP-IV produced a toluidine form for EP-4OCB-MA, and that the enzyme reaction was inhibited by the addition of an inhibitor (Figure 5). Here, Figure 5 shows LC / MS analysis of the enzymatic reaction of EP-4OCB-MA (100 μM): mass chromatograms of EP-4OCB-MA and product compounds after incubation in PBS (pH 7.4) (1% DMSO as a co-solvent) for 12 hours at 37°C in the presence or absence of DPP-IV (>0.1 U / mL) and in the presence or absence of sitagliptin (200 μM).

[0092] [Example 3] Evaluation of cell membrane permeability, cell selectivity and cytotoxicity by CCK-8 assay Next, we performed a CCK-8 assay using DPP-IV high / low expressing cell lines. First, the results of EP-4OCB-FMA are shown below. When cell viability was evaluated 24 hours after administration of EP-4OCB-FMA, a concentration-dependent decrease in cell viability was confirmed in H226 cells, HepG2 cells, and Caco-2 cells, which are high DPP-IV expressing cells. The results are shown in Figure 6. Figure 6 shows cell viability after 24 hours of EP-4OCB-FMA treatment with or without CCK8 and sitagliptin. Mean values ​​± standard deviation of concentrations compared to untreated are shown (n=3 biological replicates).

[0093] On the other hand, it was revealed that the cell viability of H460 cells, which express DPP-IV, was hardly decreased even at a high concentration of 50 μM. Furthermore, the viability of the cells with high DPP-IV expression was significantly restored by administration of the inhibitor sitagliptin, suggesting that the cytotoxicity of EP-4OCB-FMA is DPP-IV dependent. This is thought to be due to the fact that the compound has low cell membrane permeability before the enzyme reaction, but its hydrophobicity increases after the enzyme reaction, improving cell membrane permeability.

[0094] In addition, when the same evaluation was performed on H226 cells, which highly express DPP-IV, 3 hours after administration of EP-4OCB-FMA, a time-dependent decrease in cell viability was confirmed. The results are shown in Figure 7. 7 shows cell viability measurements upon EP-4OCB-FMA treatment with or without CCK8 and sitagliptin for 3 or 24 hours. Mean values ​​± standard deviation of concentrations compared to untreated are shown (n=3 biological replicates). The lack of difference in cell viability between the 25 μM and 50 μM administration groups can be explained by the assumption that EP-4OCB-FMA is gradually metabolized by DPP-IV on the cell membrane and is still in the process of being metabolized 3 hours after administration.

[0095] On the other hand, for the control compound EP-4OCB-MA, a concentration-dependent decrease in cell viability was confirmed regardless of whether DPP-IV was highly or lowly expressed. The results are shown in Figure 8. Figure 8 shows cell viability after 24 hours of EP-4OCB-MA treatment with or without CCK8 and sitagliptin. Mean values ​​± standard deviation of concentrations compared to untreated are shown (n=3 biological replicates). Considering that cell viability was not restored even with the addition of the inhibitor sitagliptin, it was suggested that toxicity was exerted in a DPP-IV-independent manner.

[0096] [Example 4] Evaluation of selectivity and cellular uptake in cultured cell lines When evaluating the extent to which BNCT drugs accumulate in cells or tumor tissues, boron quantification methods using inorganic elemental analysis such as ICP-MS (inductively coupled plasma mass spectrometry) and ICP-AES (inductively coupled plasma atomic emission spectrometry) are widely used in both in vitro and in vivo systems. Among these, MP-AES (microwave nitrogen plasma atomic emission spectrometry) is a device that can safely and easily quantify boron without using expensive or flammable gases. From the above examples, it is expected that EP-4OCB-FMA selectively accumulates in cells with high DPP-IV expression due to changes in cell membrane permeability before and after the enzyme reaction. To evaluate this in more detail, the intracellular boron concentration 3 hours after administration of EP-4OCB-FMA was quantified using MP-AES. The protocol used is shown in Figure 9. Based on the results of the CCK-8 assay in H226 cells, it was considered that 3 hours after administration of 10 μM was appropriate as a condition under which cells would not die, and the intracellular boron concentration was quantified. The results of the quantification of extracellular boron are shown in Figure 10. H226 cells were incubated with EP-4OCB-FMA for 3 h without / with a DPP-IV inhibitor (sitagliptin). For blank evaluation, cells were not seeded and only drug-containing medium was added. Results are presented as mean ± standard deviation (n = 3 biological replicates).

[0097] Three hours after administration of EP-4OCB-FMA, the boron concentration in the extracellular fluid was quantified, and it was confirmed that the boron concentration in the drug-administered group was lower than that in the blank group, in which only the drug was administered without cells. In addition, in the presence of the DPP-IV inhibitor sigatgliptin, there was almost no decrease in the boron concentration in the extracellular fluid, suggesting that EP-4OCB-FMA is first taken up into cells after undergoing an enzymatic reaction with DPP-IV on the cell membrane.

[0098] Furthermore, the intracellular boron concentration was quantified, and EP-4OCB-FMA increased the boron concentration by 10 6 It was revealed that boron accumulated in the cells at a high concentration of 0.27 μg per cell (Figure 11). The test conditions are as follows. Figure 11(a): Cellular uptake of EP-4OCB-FMA. H226 cells were incubated with EP-4OCB-FMA for 3 h with / without a DPP-IV inhibitor (sitagliptin). Results are shown as mean ± standard deviation (n = 3 biological replicates). **P < 0.001 (Student's t test). Figure 11(b): Intracellular retention of EP-4OCB-FMA. Cells were incubated with EP-4OCB-FMA for 3 h without / with further incubation in fresh medium without EP-4OCB-FMA. Results without further incubation are the same as in (a). Results are shown as mean ± standard deviation (n=3 biological replicates).

[0099] Furthermore, the addition of the DPP-IV inhibitor sitagliptin reduced the intracellular boron concentration to about one-quarter, revealing DPP-IV-dependent intracellular accumulation (see Figure 12(a)). The T / N (Tumor / Normal) ratio, which is said to be the benchmark for performing BNCT, is said to be 3, suggesting that EP-4OCB-FMA is a promising BNCT drug. In addition, the drug-containing medium was removed, replaced with fresh medium, and incubated for 30 minutes as a wash procedure. The intracellular boron concentration was then quantified in the same manner. The concentration remained almost unchanged from that before the wash procedure (see Figure 11(b)). Thus, it was suggested that EP-4OCB-FMA has excellent intracellular retention.

[0100] [Example 5] Evaluation of the reactivity of azaquinone methide intermediates with nucleophilic groups In the drug strategy of EP-4OCB-FMA, the azaquinone methide intermediate generated after the enzyme reaction with DPP-IV reacts with intracellular nucleophiles, thereby acquiring intracellular retention. To confirm that this function works, in vitro studies were performed using purified enzyme. Specifically, by reacting EP-4OCB-FMA with purified DPP-IV enzyme in the presence of L-cysteine ​​and glutathione, we evaluated using LC / MS whether these nucleophiles react with the azaquinone methide intermediate. The protocol used was the same as that used in confirming the reactivity of EP-4OCB-FMA with the purified enzyme in Example 2, with the addition of the following procedure. In experiments evaluating the reaction of azaquinone methides with nucleophiles (GSH, l-Cys), 0.1 M HEPES buffer (pH 7.4) containing 5 mM of each nucleophile was prepared and used.

[0101] The results are shown below (Figure 12). The purified enzyme was incubated in the presence of 5 mM cysteine, and reactant analysis was performed after 12 hours. MS peaks of a compound thought to be the nucleophilic addition of cysteine ​​to the azaquinone methide intermediate were detected. In addition, the MS peak of the water adduct detected at the same time was significantly lower in intensity than when cysteine ​​was not added, suggesting that water molecules and cysteine ​​compete with each other to react with the azaquinone methide intermediate. Furthermore, when cysteine ​​was not added, an MS peak at m / z = 599 was detected. Based on this value and the shape of the isotopic peak derived from carborane, it is presumed to be derived from the following compound; however, this signal was hardly detected in the presence of cysteine.

[0102] TIFF0007628340000009.tif37158

[0103] In the presence of 5 mM glutathione, more water adducts were formed than in the case of cysteine, probably because the reactivity of the SH group of glutathione is lower than that of cysteine. However, MS signals that were thought to be glutathione adducts were still detected. These results demonstrated that the azaquinone methide intermediate reacts with intracellular nucleophiles such as cysteine ​​and glutathione to form a covalent bond, suggesting that the boron drug may be trapped by intracellular nucleophiles as designed.

[0104] FIG. 12 shows LC / MS analysis of the enzymatic reaction of EP-4OCB-FMA (100 μM): Mass chromatograms of EP-4OCB-FMA and product compounds after incubation in PBS (pH 7.4) (1% DMSO as a co-solvent) for 12 hours at 37° C. in the presence or absence of DPP-IV (>0.1 U / mL).

[0105] [Synthesis Example 3] EP-4ACB-FMA (compound 36), a compound of the present invention in which a carborane moiety and a benzene ring moiety are linked via an alkyl group, was synthesized according to the following synthesis scheme 4. Details of each reaction step are described below.

[0106] Synthetic Scheme 4 TIFF0007628340000010.tif176162

[0107] (1) Synthesis of compound 27 ([3-(trimethylsilyl)prop-2-ynyl]-o-carborane) o-Carborane (689 mg, 4.78 mmol) was dissolved in 15 mL of dry THF. After cooling to -20 °C, a 1.6 M solution of n-BuLi in hexane (3.6 mL, 5.8 mmol) was added dropwise to the solution. After stirring at -20 °C for 30 min under argon atmosphere, a solution of 3-bromo-1-(trimethylsilyl)-1-propyne (1.0 g, 5.2 mmol) in 2 mL of dry THF was added dropwise. After stirring at -20 °C, the reaction solution was allowed to warm to room temperature. After stirring for 2.5 h, the reaction was quenched with H2O. The mixture was concentrated under reduced pressure. The residue was diluted with AcOEt and brine, and the organic layer was extracted, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by MPLC (OH silica gel, AcOEt / hexane) to give a pale yellow oil (888 mg, 3.49 mmol, 73%). 1 H-NMR (400 MHz, CDCl3) δ 3.93 (s, 1H), 3.20 (s, 2H), 0.18 (s, 9H), 1.40-3.20 (br, 10H).

[0108] (2) Synthesis of compound 28 (prop-2-ynyl-o-carborane) Compound 27 (782 mg, 3.07 mmol) was dissolved in 7 mL of dry THF, followed by the addition of TBAF (~1 M in THF) (3.38 mL, 3.38 mmol) and acetic acid (229 μL, 4.00 mmol). The solution was stirred at room temperature under argon for 1 h. The solvent was removed by evaporation. AcOEt was added to the residue and the organic layer was washed with brine. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by MPLC (OH silica gel, AcOEt / hexane) to give a clear solid (393 mg, 2.16 mmol, 70%). 1 H-NMR (400 MHz, CDCl3) δ 4.01 (s, 1H), 3.21 (d, J = 2.7 Hz, 2H), 2.37 (t, J = 2.7 Hz, 1H), 1.40-3.20 (br, 10H). 13 C-NMR (101 MHz, CDCl3) δ 76.35, 75.01, 69.65, 59.61, 28.31.

[0109] (3) Synthesis of compound 29 2-Amino-5-iodobenzoic acid (2.0 g, 4.35 mmol) was dissolved in 15 mL of dry THF, followed by the dropwise addition of LiAlH4 (2.5 M in THF solution) (6 mL, 15 mmol) at 0°C. After warming to room temperature, the solution was stirred at room temperature for 4 h. After carefully quenching the reaction with AcOEt, the solvent was diluted with H2O. The mixture was concentrated to remove THF and filtered through Celite®. After washing the precipitate with AcOEt, the filtrate was extracted with AcOEt, and the organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by MPLC (OH silica gel, AcOEt / hexane) to give a yellow powder (707 mg, 2.84 mmol, 38%). 1 H-NMR (400 MHz, MeOH-d4) δ 7.38 (d, J = 2.3 Hz, 1H), 7.30 (dd, J = 8.5, 2.3 Hz, 1H), 6.53 (d, J = 8.5 Hz, 1H), 4.49 (s, 2H).

[0110] (4) Synthesis of compound 30 Compound 29 (601 mg, 2.41 mmol), TBSCl (591 mg, 3.92 mmol) and imidazole (399 mg, 5.86 mmol) were added in 10 mL of dry CHCl. ​​The solution was stirred at room temperature under argon atmosphere for 2 h. AcOEt was added to the solvent and the organic layer was washed with water. The organic layer was dried over NaSO and concentrated under reduced pressure. The residue was purified by MPLC (OH silica gel, AcOEt / hexane) to give a yellow oil (855 mg, 2.35 mmol, 98%). 1 H-NMR (400 MHz, CDCl3) δ 7.34 (dd, J = 8.5, 2.3 Hz, 1H), 7.31 (d, J = 2.3 Hz, 1H), 6.44 (d, J = 8.5 Hz, 1H), 4.60 (s, 2H), 4.22 (br, 2H), 0.89 (s, 9H), 0.07 (s, 6H).

[0111] (5) Synthesis of Compound 31 Compound 30 (582 mg, 1.60 mmol) and compound 20 (340 mg, 1.87 mmol) were dissolved in 5 mL of DIEA, and then copper(I) iodide (33 mg, 0.17 mmol) was added. After degassing by freeze-pump-thaw cycles, PdCl2(PPh3)2 (56 mg, 0.080 mmol) was added to the solution. The reaction solution was stirred at 70 °C under argon atmosphere for 6 h. AcOEt was added to the solvent, and the organic layer was washed with water and brine. The organic layer was dried over Na2SO4 and concentrated under low pressure. The residue was purified by MPLC (OH silica gel, AcOEt / hexane) to give a yellow solid (370 mg, 0.877 mmol, 55%). 1 H-NMR (400 MHz, CDCl3) δ 7.15 (dd, J = 8.2, 1.8 Hz, 1H), 7.08 (d, J = 1.8 Hz, 1H), 6.58 (d, J = 8.2 Hz, 1H), 4.63 (s, 2H), 4.43 (s, 2H), 4.04 (s, 1H), 3.38 (s, 2H), 1.40-3.20 (br, 10H), 0.90 (s, 9H), 0.08 (s, 6H). 13 C-NMR (101 MHz, CDCl3) δ 147.27, 132.51, 131.95, 124.98, 115.46, 110.00, 87.28, 79.17, 71.23, 64.56, 59.58, 29.38, 25.92, 18.31, -5.17

[0112] (6) Synthesis of compound 32 Compound 31 (321 mg, 0.769 mmol) was dissolved in 10 mL of methanol, followed by the addition of a small amount of Pd / C (10%, 55% water). After stirring at room temperature under H2 for 3 h, the reaction mixture was filtered through Celite® and the filtrate was concentrated under reduced pressure. The residue was purified by MPLC (OH silica gel, AcOEt / hexane) to give a yellow oil (321 mg, 0.761 mmol, 99%). 1H-NMR (400 MHz, CDCl3) δ 6.85 (dd, J = 7.8, 1.8 Hz, 1H), 6.79 (d, J = 2.3 Hz, 1H), 6.60 (d, J = 8.2 Hz, 1H), 4.65 (s, 2H), 3.51 (s, 1H), 2.47 (t, J = 7.3 Hz, 2H), 2.14-2.19 (m, 2H), 1.70-1.74 (m, 2H), 1.40-3.20 (br, 10H), 0.90 (s, 9H), 0.07 (s, 6H). 13 C-NMR (101 MHz, CDCl3) δ 144.47, 129.64, 128.45, 128.35, 125.54, 116.03, 75.38, 64.90, 61.19, 37.44, 34.11, 31.14, 25.97, 18.36, -5.11.

[0113] (7) Synthesis of compound 33 Compound 32 (139 mg, 0.330 mmol) and N-Boc-E(OtBu)-P-OH (171 mg, 0.430 mmol) were dissolved in 2 mL of dry THF, followed by the addition of HATU (377 mg, 0.991 mmol) and DIEA (173 μL, 0.990 mmol). The solution was stirred at room temperature under argon for 3 h. The solvent was removed by evaporation. AcOEt was added to the residue and the organic layer was washed with brine. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by MPLC (OH silica gel, AcOEt / hexane) to give a white powder (209 mg, 0.260 mmol, 79%). 1 H-NMR (400 MHz, CDCl3) δ 8.99 (s, 1H), 7.95 (d, J = 8.2 Hz, 1H), 7.01 (dd, J = 8.2, 1.8 Hz, 1H), 6.95 (d, J = 1.8 Hz, 1H), 5.23 (d, J = 8.7 Hz, 1H), 4.79 (d, HBn, J gem = 12.8 Hz, 1 H), 4.61 (d, HBn, J gem= 12.8 Hz, 1H), 4.51-4.56 (m, 2H), 3.80 (m, 2H), 3.52 (s, 1H), 2.55 (t, J = 7.1 Hz, 2H), 2.30-2.42 (m, 2H), 2.00-2.28 (m, 6H), 1.69-1.80 (m, 4H), 1.44 (s, 9H), 1.43 (s, 9H), 1.40-3.20 (br, 10H), 0.92 (s, 9H), 0.12 (s, 3H), 0.07 (s, 3H). 13 C-NMR (101 MHz, CDCl3) δ 172.23, 171.81, 169.44, 155.69, 136.04, 135.12, 130.47, 128.12, 127.44, 122.34, 80.62, 79.76, 75.16, 64.34, 61.35, 61.07, 51.20, 47.44, 37.32, 34.38, 31.00, 30.76, 28.72, 28.42, 28.18, 28.01, 25.94, 25.25, 18.35, -5.07

[0114] (8) Synthesis of compound 34 Compound 33 (182 mg, 0.226 mmol) was dissolved in 3 mL of dry THF, followed by the addition of TBAF (~1 M in THF) (726 μL, 0.726 mmol) and acetic acid (27.7 μL, 0.484 mmol). The solution was stirred at room temperature under argon for 40 min and the solvent was removed by evaporation. AcOEt was added to the residue and the organic layer was washed with water. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by MPLC (OH silica gel, AcOEt / hexane) to give a white solid (132 mg, 0.191 mmol, 85%). 1H-NMR (400 MHz, CDCl3) δ 9.29 (s, 1H), 7.89 (d, J = 8.2 Hz, 1H), 7.05 (dd, J = 8.2, 1.8 Hz, 1H), 6.96 (d, J = 1.8 Hz, 1H), 5.32 (d, J = 8.7 Hz, 1H), 4.73 (dd, J = 7.8, 2.7 Hz, 1H), 4.61 (s, 2H), 4.56 (td, J = 8.7, 3.7 Hz, 1H), 3.87 (brs, 1H), 3.73-3.83 (m, 2H), 3.54 (s, 1H), 2.53 (t, J = 7.3 Hz, 2H), 2.23-2.40 (m, 3H), 2.05-2.19 (m, 6H), 1.71-1.85 (m, 3H), 1.44 (s, 9H), 1.41 (s, 9H), 1.40-3.20 (br, 10H). 13 C-NMR (101 MHz, CDCl3) δ 173.25, 172.62, 169.69, 155.57, 136.72, 135.19, 130.78,

[0115] (9) Synthesis of Compound 35 Compound 34 (112 mg, 0.162 mmol) was dissolved in 6 mL of dry CHCl, then Deoxo-Fluor® (59.7 μL, 0.324 mmol) solution was added dropwise to 4 mL of dry CHCl at 0° C. The reaction solution was stirred at room temperature under argon atmosphere for 1 h. CHCl was added to the reaction solution and washed with saturated aqueous NaHCO. The organic layer was dried over NaSO and concentrated under reduced pressure. The residue was purified by MPLC (OH silica gel, AcOEt / hexane) to give a clear solid (88 mg, 0.127 mmol, 78%). 1H-NMR (400 MHz, CDCl3) δ 8.95 (s, 1H), 7.83 (d, J = 8.7 Hz, 1H), 7.13 (d, J = 8.2 Hz, 1H), 7.08 (s, 1H), 5.30-5.42 (m, 3 H), 4.78 (dd, J = 8.0, 2.1 Hz, 1H), 4.55 (td, J = 9.4, 3.5 Hz, 1H), 3.75 (dd, J = 7.8, 5.0 Hz, 2H), 3.54 (s, 1H), 2.48-2.58 (m, 3H), 2.28-2.42 (m, 2H), 2.16-2.20 (m, 2H), 1.94-2.11 (m, 3H), 1.71-1.81 (m, 4H), 1.44 (s, 9H), 1.43 (s, 9H), 1.40-3.20 (br, 10H). 13 C-NMR (101 MHz, CDCl3) δ 173.33, 172.19, 169.37, 155.64, 137.15, 134.75, 134.72, 129.83, 129.80, 129.30, 129.24, 127.37, 127.21, 123.68, 83.32, 81.68, 80.83, 79.97, 77.46, 77.15, 76.83, 75.03, 61.36, 60.84, 51.18, 47.67, 37.43, 34.35, 31.05, 30.76, 28.41, 28.14, 28.06, 27.18, 25.31.

[0116] (10) Synthesis of Compound 36 Compound 35 (53 mg, 0.077 mmol) was dissolved in 1 mL of TFA. The solution was stirred at room temperature for 10 min, and the reaction solution was diluted with CHCl and concentrated by evaporation. The residue was purified by HPLC (eluent: A: H0, 0.1% TFA (v / v), eluent: B: CHCN / H0 = 80 / 20, 0.1% TFA (v / v) to give a white powder (24 mg, 0.045 mmol, 58%). 1H-NMR (400 MHz, MeOH-D4) δ 7.19-7.30 (m, 3H), 5.24-5.49 (m, 2H), 4.63 (dd, J = 8.2, 5.5 Hz, 1H), 4.52 (s, 1H), 4.38 (dd, J = 7.4, 4.9 Hz, 1H), 3.68-3.80 (m, 2H), 2.55-2.64 (m, 4H), 1.98-2.42 (m, 8H), 1.76-1.84 (m, 2H), 1.40-3.20 (br, 10H). 13 C-NMR (101 MHz, Acetonitrile-d3) δ 175.17, 170.52, 167.60, 139.29, 133.30, 133.26, 130.64, 130.48, 129.36, 128.84, 128.77, 125.27, 82.56, 80.96, 76.29, 62.37, 61.00, 51.47, 47.50, 36.79, 33.85, 30.67, 29.00, 24.96, 24.88

Claims

1. A compound represented by the following general formula (I) or a salt thereof. (In the formula, X is a fluorine atom, an ester group (-OC(=O)-R'), a carbonate group (-OCO ２ -R'), carbamate group (-OCONH-R'), phosphoric acid and its ester group (-OP(=O)(-OR')(-OR''), and sulfuric acid and its ester group (-OSO ２ —OR′), wherein R′, R″ are each independently selected from a substituted or unsubstituted alkyl group, or a substituted or unsubstituted aryl group; Y is an enzyme recognition site, a part of which is cleaved by the enzymatic activity of a glycosidase or peptidase to induce the formation of a quinone methide; Y is -NH-CO-L or -NH-L'; wherein L together with the C=O to which it is attached constitutes an amino acid residue or a peptide; L' is a saccharide moiety having a self-cleavable linker, an amino acid residue having a self-cleavable linker, or a peptide; Here, the partial structure of a saccharide is a structure obtained by removing one hydroxyl group from a saccharide, R １ and R ２ are each independently selected from a hydrogen atom or a monovalent substituent; R ３ are a hydrogen atom or 1 to 3 identical or different monovalent substituents present on a benzene ring; Z represents a single bond or a linking group: B is, １０ represents a group containing B, which is a group derived from a boron cluster.

2. The compound or salt thereof according to claim 1, wherein B is a group derived from a compound having at least one boron atom in the molecule.

3. The compound or salt thereof according to claim 1 or 2, wherein the boron cluster has a polyhedral structure.

4. The compound or salt thereof according to any one of claims 1 to 3, wherein B is a group derived from closododecaborate, closodocarborane, nidocarborane, bisdicarbollide metal complex, GB10, 1,2-dicarbacloso-dodecaborane, 1,7-dicarba-closo-dodecaborane, 1,12-dicarba-closo-dodecaborane, dicarba-closo-decaborane, or sulfur-substituted undecahydrododecaborate.

5. The linking group is an alkylene group (provided that the alkylene group has one or more —CH ２ The compound or salt thereof according to any one of claims 1 to 4, wherein - may be substituted with -O-, -S-, -NH-, or -CO-, arylene (including heteroarylene), cycloalkylene, an alkoxyl group, a polyethylene glycol chain, and a group formed by arbitrarily bonding two or more groups selected from these groups.

6. The compound or salt thereof according to any one of claims 1 to 5, wherein the saccharide is 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, or β-D-N-acetylgalactosamine.

7. In the general formula (I), -Y is -C(R １ ) (R ２ 7. The compound or salt thereof according to claim 1, wherein X is bonded to the ortho- or para-position of the benzene ring.

8. The compound or salt thereof according to any one of claims 1 to 7, wherein Y has a structure selected from the following:

9. The compound or salt thereof according to any one of claims 1 to 8, wherein X is a fluorine atom or an ester group (-OC(=O)-R').

10. R １ and R ２ are each independently selected from a hydrogen atom or a fluorine atom, or a salt thereof.

11. R ３ The monovalent substituent is an alkyl group, an alkoxycarbonyl group (-C(=O)-OR'), a nitro group, an amino group, a hydroxyl group, an alkylamino group (-NHR', -NR' ２ The compound or salt thereof according to any one of claims 1 to 10, wherein R' is a substituted or unsubstituted alkyl group, or a substituted or unsubstituted aryl group, and when there are two or more R', they may be the same or different.

12. R ３ The compound or salt thereof according to claim 11, wherein the monovalent substituent is an alkyl group or an alkoxy group.

13. R ３ The compound or salt thereof according to claim 11, wherein the monovalent substituent of the formula (I) is a halogen atom.

14. R ３ one or more of the monovalent substituents of R is an alkyl group or an alkoxy group; ３ The compound or salt thereof according to any one of claims 11 to 13, wherein one or more of the monovalent substituents of the formula (I) is a halogen atom.

15. R ３ The compound or salt thereof according to any one of claims 1 to 10, wherein all of are hydrogen atoms.

16. A pharmaceutical composition comprising a compound according to any one of claims 1 to 15 or a pharma- ceutically acceptable salt thereof.

17. The pharmaceutical composition according to claim 16, for use in boron neutron capture therapy.

18. The pharmaceutical composition according to claim 17, which can accumulate in cancer cells by acting selectively on the cells through a cancer cell-specific enzymatic activity.

19. 19. The pharmaceutical composition of claim 18, wherein the enzyme is a peptidase or a glycosidase.

20. 1. A method for diagnosing, treating, or diagnosing and treating a disease or a condition that may lead to a disease, comprising: (A) administering to a subject (excluding humans) having or suspected of having a disease or condition a pharmaceutical composition comprising a compound according to any one of claims 1 to 15 or a pharma- ceutical acceptable salt thereof; and (B) localized to a target tissue of the subject. １０ Irradiating the B atoms with neutrons, thereby performing boron neutron capture therapy on the target tissue. The method comprising:

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