Novel boron-containing drugs

Abstract

Provided is a boron drug that is for boron neutron capture therapy and that is characterized by containing a compound represented by formula (I) or (II) [in the formulae, C represents a carbon atom, L1, L2, L3, and L4 each independently represent a divalent group that functions as a spacer, X represents a group that binds with albumin, Y represents a group including 10B, and Z represents a group that binds with a folate receptor].

Description

[Technical Field]

[0001] The present invention relates to a boron agent for boron neutron capture therapy, and an imaging agent for PET (positron emission tomography) for predicting the effects of this boron agent for boron neutron capture therapy. [Background technology]

[0002] Boron neutron capture therapy (BNCT) uses a powerful particle beam produced by the nuclear reaction of low-energy thermal and epithermal neutrons with boron, and is attracting attention as the ultimate minimally invasive treatment method. BPA, the precursor drug for BNCT, was approved in March 2020 along with a domestically produced small accelerator for BNCT for the treatment of "unresectable locally advanced or locally recurrent head and neck cancer," and insurance coverage for treatment began in June. BPA is currently the only boron-based drug used in BNCT, but approximately 40% of patients eligible for BNCT treatment are insensitive to BPA, and many patients are unable to receive BNCT, making the development of new drugs an urgent need.

[0003] The inventors have so far developed candidate boron-based drugs to replace BPA, including a conjugate (Patent Document 1) in which a boron cluster and albumin are linked by maleimide, and a folic acid derivative (Patent Document 2) that targets folic acid receptors, which are highly expressed in many cancer cells. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] International Publication No. 2017 / 026276 [Patent Document 2] Japanese Patent Publication No. 2019-38778 [Overview of the project] [Problems that the invention aims to solve]

[0005] The boron-containing drug developed by the present inventors is expected to have a high antitumor effect, but the development of new boron-containing drugs is desired for more effective cancer treatment. This invention was made against this backdrop and aims to provide a novel boron-containing drug for BNCT.

[0006] Furthermore, before administering the above BNCT drug to the patient, this BNCT drug 18 By administering a substance labeled with F and performing a PET scan, it is possible to predict the effects of the drug. This invention also aims to provide such a drug for PET scans. [Means for solving the problem]

[0007] The inventors, through diligent research to solve the above problems, have discovered that the antitumor effect of boron-containing drugs can be improved by binding boron to an albumin ligand. Furthermore, they have found that the antitumor effect of boron-containing drugs can be further improved by binding a folic acid receptor recognition site to these boron-containing drugs. This invention was completed based on these findings.

[0008] In other words, the present invention provides the following (1) to (12). (1) The following formula (I) or (II) [ka] [In the formula, C represents carbon atom, L 1 , L 2 , L 3 , and L 4 Each of these independently represents a divalent group that functions as a spacer, X represents the group that binds to albumin, and Y represents, 10 A boron agent for boron neutron capture therapy, characterized by containing a compound represented by [B represents a group containing a folate receptor, and Z represents a group that binds to a folate receptor].

[0009] (2) L in equations (I) and (II) 1 , L 2 , L3 and L 4 is an alkylene group (however, one or more -CH2- of the alkylene group may be substituted with -O-, -S-, -NH-, or -CO-), and the boron agent for boron neutron capture therapy according to (1).

[0010] (3) In formulas (I) and (II), X is represented by the following formulas (A) to (C)

Chemical formula

[0011] (4) In formulas (I) and (II), Y is a group derived from a boron cluster, and the boron agent for boron neutron capture therapy according to any one of (1) to (3).

[0012] (5) In formula (II), Z is represented by the following formula (D)

Chemical formula

[0013] (6) The compounds represented by formulas (I) and (II) are respectively the following formulas (Ia) and (IIa)

Chemical formula

[0014] (7) The following formula (III) or (IV)

Chemical formula

[0015] (8) L in equations (III) and (IV) 5 , L 6 , L 8 , L 9 and L 10 However, it is an alkylene group (wherein one or more -CH2- groups of the alkylene group may be substituted with -O-, -S-, -NH-, or -CO-), L 7 The PET imaging agent according to (7), characterized in that it is an alkylene group (however, one or more -CH2- groups of the alkylene group may be substituted with -O-, -S-, -NH-, or -CO-, and one -CH2- group of the alkylene group may be substituted with a divalent group formed by a click reaction).

[0016] (9) X in equations (III) and (IV) is given by equations (A) to (C) below. [ka] (In the formula, * represents a bonding site, and R represents a hydrogen atom, fluorine atom, chlorine atom, bromine atom, or iodine atom.) The PET imaging agent according to (7) or (8), characterized in that it is a group represented by .

[0017] (10) The PET imaging agent according to any one of (7) to (9), characterized in that Y in formulas (III) and (IV) is a group derived from a boron cluster.

[0018] (11) Z in equation (IV) is given by equation (D) below [ka] (In the formula, * represents a bonding site.) A PET imaging agent according to any one of (7) to (10), characterized in that it is a group represented by (1).

[0019] (12) The compound represented by formula (IV) is the following formula (IVa) [ka] The PET imaging agent according to (7), characterized in that it is a compound represented by .

[0020] This specification includes the content described in the specifications and / or drawings of the Japanese patent applications, Japanese Patent Application No. 2021-039885 and Japanese Patent Application No. 2021-096432, which form the basis of the priority of this application. [Effects of the Invention]

[0021] This invention provides a novel boron-containing drug for use in BNCT, and a novel PET imaging agent for predicting the effects of this boron-containing drug. [Brief explanation of the drawing]

[0022] [Figure 1] A diagram showing the expression levels of folate receptor (FRα) in various cancer cells. [Figure 2] This figure shows the quantitative results of folate receptor (FRα) levels obtained by flow cytometry. [Figure 3]Figure showing the thermal neutron dose-dependent antitumor effect of BNCT. (A) Human brain tumor cells U87MG (FRα(+)), (B) Human lung cancer cells A549 (FRα(-)). Drug 10B concentration 25 ppm, cultured for 3 hours, drug removed, and irradiated. [Figure 4] This figure shows (A) pharmacokinetics (7.5 mg (10B) / kg), (B) antitumor effect, and (C) body weight change of PBC-IP albumin complex and BPA in mice subcutaneously transplanted with human brain tumor cells U87MG (FRα(+)). [Figure 5] A diagram showing the selective accumulation ability of PBC-IP (A) and BPA (B) in various cell types. [Figure 6] Table showing the distribution of boron-containing drugs in F98 and C6 osthotopic brain tumor rats. [Figure 7] Figure 7(A) shows the efficacy of BNCT therapy with PBC-IP in a rat brain tumor model transplanted with malignant glioblastoma cells (F98 and C6). Three months after irradiation, 50% survival was observed in the PBC-IP group, and 70% survival was observed in the group treated with BPA (Figure 7(A)). [Figure 8] Brain tissue staining images of rats 100 days after BNCT (A) and untreated rats (B). [Modes for carrying out the invention]

[0023] The present invention will be described in detail below. (1) Boron-containing chemicals for BNCT The boron agent for BNCT of the present invention is of the following formula (I) or (II) [ka] It is characterized by containing a compound represented by [the formula shown].

[0024] In BNCT (Boron Neutron Capture Therapy), a neutron beam is irradiated onto a patient who has been previously administered a boron drug. The reaction between the boron drug and the neutrons generates lithium and alpha rays in the microenvironment within a single cell, thereby destroying cancer cells. In this invention, "boron drug for BNCT" refers to the boron drug used in this BNCT.

[0025] In formulas (I) and (II), X is not particularly limited as long as it is a group that binds to albumin, but a group that binds to albumin non-covalently is preferred. This is because if the boron drug is covalently bound to albumin, the boron drug may not be released within the tumor, and the drug may not exert its full efficacy. Many groups that bind to albumin or non-covalently are known, and in the present invention, such known groups can be appropriately selected and used. Specific examples of groups that bind to albumin non-covalently include the iodobutyrate group shown in formula (A) below, the group contained in Evans Blue shown in formula (B) below, and the group shown in formula (C) below. [ka]

[0026] In equations (I) and (II), Y is: 10 The group is not particularly limited as long as it contains B, and may be a group derived from a compound that has one boron atom in its molecule, such as BPA, but a group derived from a boron cluster is preferred. The boron cluster can be any polyhedral structure that can be used in boron neutron capture therapy, for example, clothododecaborate ([B 12 H 12 ] 2- ), ionic clothocarborane ([CB 11 H 12 ] - ), lipid-soluble clothocarborane ([C2B 10 H 12 ]), Nidocarborane ([C2B9H 11 ] - ), bis-dicarboridol metal complex ([(C2B9H 11 )2M]), GB10([B 10 H 12 ] 2-Examples include the following. Boron clusters are preferably water-soluble boron clusters such as clothododecaborate, ionic clothocarborane, nidocarborane, and GB10. All boron atoms contained in the boron cluster are 10 B is also acceptable, but only a part of it 10 B may also be used. In this specification, the expression "derived group" is used, as in "group derived from boron cluster," which means a group derived, for example, by removing one hydrogen atom from a boron cluster.

[0027] In formula (II), Z is not particularly limited as long as it is a group that binds to the folate receptor. Many groups that bind to the folate receptor are known, and in the present invention, such known groups can be appropriately selected and used. Specific examples of groups that bind to the folate receptor include the groups contained in folate shown in formula (D) below. [ka]

[0028] By using a compound with a group that binds to the folate receptor as a boron drug, it becomes possible to introduce the boron drug into cancer cells via the folate receptor, which is highly expressed in many cancer cells. Furthermore, while BPA, a known boron drug, is taken up by cells via an amino acid transporter called LAT-1, the expression of LAT-1 is reduced in BPA-insensitive cancers. Compounds with a group that binds to the folate receptor are taken up by cancer cells without the involvement of LAT-1, and are therefore effective against BPA-insensitive cancers as well.

[0029] The group that binds to the folate receptor is for uptake by cancer cells, but as shown in Example 6, having such a group improves its binding affinity to albumin. As a result, blood retention improves, and delivery to cancer cells also improves.

[0030] L in equations (I) and (II) 1, L 2 , L 3 , and L 4 The spacer is not particularly limited as long as it is a divalent group, but it is preferably a linear divalent group, and more preferably an alkylene group. However, one or more -CH2- groups of the alkylene group may be substituted with -O-, -S-, -NH-, or -CO-. The number of carbon atoms in the alkylene group is determined by the number of carbon atoms in the group that binds to albumin. 10 The distance between two groups containing B, or the group that binds to albumin. 10 The number of groups is not particularly limited as long as it is sufficient to ensure a adequate distance between the three parties: the group containing B and the group that binds to the folate receptor, but L 1 For L, 5 to 20 is preferred, 10 to 15 is more preferred. 2 For L, 3 to 15 is preferred, 5 to 10 is more preferred, 3 For L, 5 to 20 is preferred, 10 to 15 is more preferred. 4 For this, 2 to 10 is preferred, and 3 to 8 is more preferred. Furthermore, even when the -CH2- in the alkylene group is substituted with -O-, -S-, or -NH-, these groups are considered to have one carbon atom and are included in the "number of carbon atoms in the alkylene group" as described above. 1 Specific examples include the -CH2-CH2-CH2-CH2-CH2-CH2-NH-CH2-CH2-O-CH2-CH2-O- and divalent groups of similar length found in the compound represented by formula (Ia), L 2 Specific examples include the -CH2-CH2-CH2-CH2-CH2-CH2-NH-CO- group and divalent groups of similar length found in the compound represented by formula (IIa), L 3 Specific examples include the -NH-CO-CH2-CH2-CO-NH-CH2-CH2-O-CH2-CH2-O- and divalent groups of similar length found in the compound represented by formula (IIa), L 4 Specific examples include the -CH2-CH2-CH2-CH2- group and divalent groups of similar length found in the compound represented by formula (IIa).

[0031] Compounds represented by formula (I) or (II) can be synthesized according to the methods described in the examples, or by methods that appropriately modify or alter those methods with reference to those descriptions. For example, a compound having a group that binds to albumin and 10 By bonding a compound having a group containing B, the compound represented by formula (I) can be synthesized, and the compound having a group that binds to albumin 10 The compound represented by formula (II) can be synthesized by attaching a compound having a group containing B, and then attaching a compound having a group that binds to the folate receptor.

[0032] Specific examples of compounds represented by formula (I) and formula (II) include, for example, the following formulas (Ia) and (IIa), respectively. [ka] Compounds represented by the formula (Ia) can be listed below. Note that, hereafter, compounds represented by formula (Ia) may be referred to as "BC-IP," and compounds represented by formula (IIa) may be referred to as "PBC-IP."

[0033] The boron-containing drug for BNCT of the present invention is administered to humans or non-human animals. Examples of non-human animals include mice, rats, hamsters, rabbits, cats, dogs, cattle, sheep, and monkeys.

[0034] Diseases 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, and sarcoma.

[0035] While there are no particular limitations on the patients to be treated, it is preferable to target patients who are BPA-insensitive. Here, "BPA-insensitive patients" refers to those in whom the accumulation of BPA in tumor cells is low. Typically, before performing boron neutron capture therapy with BPA... 18PET diagnosis using F-BPA is performed to estimate the amount of BPA accumulation in tumor cells, and it is considered desirable to perform boron neutron capture therapy in patients whose tumor / normal tissue ratio and tumor / blood concentration ratio are 2.5 (or 3) or higher in this PET diagnosis. Accordingly, in the present invention, patients whose tumor / normal tissue ratio, tumor / blood concentration ratio, or both are less than 3 or less than 2.5 in the above PET diagnosis can be classified as "BPA-insensitive patients."

[0036] The boron agent for BNCT of the present invention can be formulated by mixing it with a pharmaceutically acceptable carrier or diluent according to known methods. The dosage form is not particularly limited and can be an injection, tablet, powder, granule, capsule, liquid, suppository, sustained-release, etc. The method of administration is also not particularly limited and can be administered orally or parenterally. Examples of parenteral administration methods include injection or infusion into the skin, intraperitoneal cavity, vein, artery, or cerebrospinal fluid. Local administration by CED (Convection Enhanced Delivery) can also be performed. The dosage varies depending on the target patient, method of administration, etc., but for example, when administering the compound represented by formula (I) or (II) as an injection to an adult, the compound represented by formula (I) or (II) can be administered in 1 to several divided doses per treatment, with a total dose of 5 to 1000 mg / kg per dose. Furthermore, before administering the boron agent for BNCT of the present invention, the PET imaging agent of the present invention, as described later, may be administered, and the dosage may be determined based on the in-body distribution and temporal changes of the compound represented by formula (III) or (IV).

[0037] The boron agent for BNCT of the present invention may be used in combination with known boron agents for BNCT (e.g., BPA, BSH). As shown in Figure 7(A), the survival time extension effect is further improved when the boron agent for BNCT of the present invention (PBC-IP) is used in combination with BPA, so BPA is preferred as the known boron agent for BNCT to be used in combination.

[0038] Since the boron-containing drug for BNCT of the present invention can bind to albumin, it can also be bound to albumin before administration and administered as an albumin complex.

[0039] (2) Imaging agents for PET The PET imaging agent of the present invention is of the following formula (III) or (IV) [ka] It is characterized by containing a compound represented by [the formula shown].

[0040] The compounds represented by formulas (III) and (IV) are: 18 Since it is labeled with F, it can emit positrons. The emitted positrons immediately combine with electrons and emit gamma rays. By measuring these gamma rays with a device used in PET, the intracellular distribution of the compounds represented by formulas (III) and (IV) can be quantitatively and temporally imaged. The compounds represented by formulas (III) and (IV) are 18 Except for being labeled with F, these compounds are similar to those represented by formulas (I) and (II), respectively. Therefore, it is possible to infer the in-vivo distribution and temporal changes of the compounds represented by formulas (I) and (II) from the in-vivo distribution and temporal changes of the compounds represented by formulas (III) and (IV). Consequently, by administering the compound represented by formula (III) or (IV) to a patient, it is possible to infer whether the patient is sensitive to the compounds represented by formulas (I) and (II), or how much of the compounds represented by formulas (I) and (II) needs to be administered to obtain an anti-cancer effect.

[0041] In equations (III) and (IV), X, Y, and Z can be exemplified by the same bases as X, Y, and Z in equations (I) and (II).

[0042] L in equations (III) and (IV) 5 , L 6 , L 7 , L 8 , L 9and L 10 The spacer is not particularly limited as long as it is a divalent group, but it is preferably a linear divalent group, and more preferably an alkylene group. However, L 5 , L 6 , L 8 , L 9 and L 10 In this case, one or more -CH2- groups of the alkylene group may be substituted with -O-, -S-, -NH-, or -CO-. Also, L 7 In this, one or more -CH2- groups of the alkylene group may be substituted with -O-, -S-, -NH-, or -CO-, and one -CH2- group of the alkylene group may be substituted with a divalent group formed by a click reaction. Here, a divalent group formed by a click reaction is, for example, a divalent group formed by the reaction of an alkyne and an azide group. Many divalent groups formed by click reactions and divalent groups formed by the reaction of alkynes and azide groups are known, and in the present invention, such known groups can be appropriately selected and used. An example of a divalent group formed by the reaction of an alkyne and an azide group is the divalent group formed by the reaction of bicyclononine contained in the compound represented by formula (IVa) with an azide group. The number of carbon atoms in the alkylene group is the number of carbon atoms in the group that binds to albumin and 10 A group containing B 18 The distance between the three F groups, or the group that binds to albumin. 10 A group containing B 18 The number of groups is not particularly limited as long as it can ensure sufficient distance between F and the folate receptor-binding group, but L 5 For L, 5 to 25 is preferred, 10 to 20 is more preferred, 6 For L, 3 to 15 is preferred, 5 to 10 is more preferred, 7 For L, 5 to 25 is preferred, and 10 to 20 is more preferred. 8 For L, 3 to 15 is preferred, 5 to 10 is more preferred, 9 For L, 3 to 20 is preferred, 5 to 15 is more preferred. 10For this, 2 to 10 is preferred, and 3 to 8 is more preferred. Furthermore, even when the -CH2- in the alkylene group is substituted with -O-, -S-, or -NH-, these groups are considered to have one carbon atom and are included in the "number of carbon atoms in the alkylene group" as described above. On the other hand, L 7 In the case where one -CH2- of the alkylene group is replaced with a divalent group formed by a click reaction, this divalent group is not included in the "number of carbon atoms of the alkylene group" as described above. 6 Specific examples include the -CO-NH-CH2-CH2-O-CH2-CH2-O- and divalent groups of similar length contained in the compound represented by formula (IVa), L 7 Specific examples include the compound represented by formula (IVa) -NH-CO-CH2-(a divalent group formed by a click reaction)-CH2-O-CO-NH-CH2-CH2-O-CH2-CH2-O-CH2-CH2- and divalent groups of similar length, L 8 Specific examples include the -CH2-CH2-CH2-CH2-CH2-CH2-NH-CO- group and divalent groups of similar length found in the compound represented by formula (IVa), L 9 Specific examples include the -NH-CO-CH2-CH2-CO-NH-CH2-CH2-CH2-CH2- contained in the compound represented by formula (IVa), and divalent groups of similar length, L 10 Specific examples include the -CH2-CH2-CH2-CH2- and divalent groups of similar length found in the compound represented by formula (IVa).

[0043] Compounds represented by formula (III) or (IV) can be synthesized according to the methods described in the examples, or by methods that appropriately modify or alter those methods with reference to those descriptions.

[0044] A specific example of a compound represented by formula (IV) is, for example, the following formula (IVa): [ka] Examples of compounds represented by [formula] can be given.

[0045] The PET imaging agent of the present invention can be formulated by mixing it with a pharmaceutically acceptable carrier or diluent according to known methods. The dosage form is not particularly limited and can be an injection, tablet, powder, granule, capsule, liquid, suppository, sustained-release, etc. The method of administration is also not particularly limited and can be administered orally or parenterally (by injection or infusion into the skin, peritoneal cavity, vein, artery, or cerebrospinal fluid, etc.). The dosage will vary depending on the target and method of administration, but for example, when administering the compound represented by formula (III) or (IV) as an injection to an adult, the compound represented by formula (III) or (IV) can be administered in an amount of 1 μg to 1000 μg / kg. [Examples]

[0046] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. [Example 1] Synthesis scheme of PBC-IP [ka] Compound 2 (Li, YX; Qiu, Z. Asian, J. Chem. 2014, 26, 3219), monobenzyl succinate (Isomura, S.; Wirs, ing, P.; Janda, KDJ Org. Chem. 2001, 66, 4115), compound 9 (Ishii, S.; Nakamura, HJ Organomet. Chem. 2018, 865, 178), and pteroylazide (Luo, J.; Smith, MD; Lantrip, DA; Wang, S.; Fuchs, PLJ Am. Chem. Soc. 1997, 119, 10004) were synthesized according to known methods.

[0047] <Synthesis of Compound 3 from Compound 2> [ka] Under an argon atmosphere, 4-iodophenylbutyric acid (4.787 g, 16.5 mmol) and N-methylmorpholine (1.91 mL, 17.3 mmol) were dissolved in dichloromethane (33 mL), and isobutyl chloroformate (2.28 mL, 17.3 mmol) was added dropwise at -15°C. After stirring at -15°C for 20 minutes, a solution of compound 2 (4.1049 g, 19.0 mmol) and N-methylmorpholine (1.81 mL, 16.5 mmol) in dichloromethane (33 mL) was added dropwise at -15°C. After addition, the mixture was stirred at room temperature for 30 minutes, then water was added to stop the reaction, and the mixture was extracted three times with dichloromethane. The organic layer was dried over magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The resulting crude product was dissolved in chloroform / methanol at 60°C, hexane was added, and the mixture was cooled to room temperature. The resulting solid was filtered and dried under reduced pressure to obtain compound 3 (6.5081 g, yield 81%) as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 7.71 (t, J = 5.4 Hz, 1H), 7.62 (d, J = 8.2 Hz, 2H), 7.00 (d, J = 8.2 Hz, 2H), 6.74 (brs, 1H), 3.00 (q, J = 6.0 Hz, 2H), 2.88 (q, J = 6.4 Hz, 2H), 2.50 (t, J = 7.3 Hz, 2H), 2.03 (t, J = 7.3 Hz, 2H), 1.75 (quint, J = 7.5 Hz, 2H), 1.36 (brs, 13H), 1.23-1.21 (m, 4H).

[0048] <Synthesis of Compound 4 from Compound 3> [ka] Under an argon atmosphere, compound 3 (6.300 g, 12.9 mmol) was dissolved in dichloromethane (65 mL), and then trifluoroacetic acid (8.0 mL) was added dropwise at 0 °C. After stirring at 0 °C for 9 hours, 1 M aqueous sodium hydroxide solution was added to stop the reaction, and the mixture was extracted three times with dichloromethane. The organic layer was dried over magnesium sulfate, filtered, and the solvent was removed under reduced pressure to quantitatively obtain compound 4 (5.446 g) as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 7.73 (d, J = 5.3 Hz, 1H), δ 7.78-7.66 (m, 2H), 7.63 (d, J = 8.2 Hz, 1H), 7.01 (d, J = 8.3 Hz, 2H), 3.01 (q, J = 6.8 Hz, 3H), 2.61 (qt, J = 7.0 Hz, 3H), 2.04 (t, J = 7.4 Hz, 1H), 1.76 (quin, J = 7.6 Hz, 2H), 1.48-1.32 (m, 4H), 1.31-1.18 (m, 4H).

[0049] <Synthesis of Compound 5 from Compound 4>

Chemical Structure

[0050] <Synthesis of Compound 6 from Compound 5> [ka] Under an argon atmosphere, compound 5 (6.513 g, 7.76 mmol) was dissolved in N,N-dimethylformamide (24 mL), and then piperidine (2.4 mL) was added at room temperature. After stirring for 60 minutes, water was added to stop the reaction, and the mixture was extracted three times with dichloromethane. The organic layer was washed with 1 M hydrochloric acid and saturated sodium bicarbonate aqueous solution, dried over magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The resulting crude product was purified by silica gel column chromatography (dichloromethane / methanol = 90:10) to obtain compound 6 (4.078 g, yield 85%) as a white solid. 1 H NMR (500 MHz, DMSO-d6) δ 7.89 (d, J = 7.5 Hz, 2H), δ 7.82 (t, J = 5.3 Hz, 1H), δ 7.78-7.66 (m, 2H), 7.62 (d, J = 8.1 Hz, 1H), 7.46-7.37 (m, 3H), 7.33 (t, J = 7.3 Hz, 2H), 7.00 (d, J = 8.2 Hz, 2H), 6.77 (d, J = 5.1 Hz, 1H), 4.35-4.20 (m, 3H), 3.98-3.88 (m, 1H), 3.13-2.96 (m, 4H), 2.89 (t, J = 5.0 Hz, 1H), 2.50-2.45 (m, 2H), 2.04 (t, J = 7.4 Hz, 1H), 1.76 (quin, J = 7.5 Hz, 2H), 1.65-1.47 (m, 2H), 1.44-1.32 (m, 15H), 1.31-1.17 (m, 6H).

[0051] <Synthesis of Compound 8 from Compound 6> [ka] Under an argon atmosphere, monobenzyl succinate (687.1 mg, 3.3 mmol) and N-methylmorpholine (363 μL, 3.30 mmol) were dissolved in dichloromethane (9.0 mL), and then isobutyl chloroformate (433 μL, 3.30 mmol) was added dropwise at -15°C. After stirring at -15°C for 20 minutes, a solution of compound 6 (1.850 g, 3.00 mmol) and N-methylmorpholine (330 μL, 3.00 mmol) in dichloromethane (9.0 mL) was added dropwise at -15°C. After addition, the mixture was stirred at room temperature for 60 minutes, then water was added to stop the reaction, and the mixture was extracted three times with dichloromethane. The organic layer was washed with saturated brine, dried over magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The crude product obtained was purified by silica gel column chromatography (dichloromethane / methanol = 95:5) to obtain a mixture containing compound 7.

[0052] The resulting mixture was dissolved in methanol (6.0 mL) and distilled water (1.5 mL), and then lithium hydroxide monohydrate (378 mg, 9.00 mmol) was added at room temperature. After stirring for 30 minutes, 1 M HCl was added to stop the reaction, and the mixture was extracted three times with dichloromethane. The organic layer was washed with saturated brine, dried over magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The resulting solid was washed with diethyl ether and dried under reduced pressure to obtain compound 8 (2.120 g, 99% yield in 2 steps) as a white solid. 1H NMR (500 MHz, DMSO-d6) δ 7.95 (d, J = 8.1 Hz, 1H), 7.78-7.71 (m, 2H), 7.61 (d, J = 8.3 Hz, 2H), 7.00 (d, J = 8.3 Hz, 2H), 6.72 (t, J = 5.5 Hz, 1H), 4.17-4.11 (m, 1H), 3.09-2.94 (m, 4H), 2.94-2.82 (m, 2H), 2.6-2.34 (m, 6H), 2.04 (d, J = 7.4 Hz, 2H), 1.76 (quin, J = 7.4 Hz, 2H), 1.64-1.41 (m, 2H), 1.41-1.29 (m, 15H), 1.29-1.14 (m, 6H).

[0053] <Synthesis of Compound 10 from Compound 8> [ka] Under an argon atmosphere, compound 8 (716.7 mg, 1.00 mmol), compound 9 (730.0 mg, 1.00 mmol), and HOBt·H2O (168.5 mg, 1.10 mmol) were dissolved in dichloromethane (6.0 mL). Then, diisopropylethylamine (209 μL, 1.20 mmol) and EDCI·HCl (210.9 mg, 1.10 mmol) were added at room temperature. After stirring for 1.5 hours, the reaction was stopped by adding saturated sodium bicarbonate aqueous solution, and the mixture was extracted three times with dichloromethane. The organic layer was washed with saturated brine, dried over magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The resulting crude product was purified by silica gel column chromatography (dichloromethane / methanol = 95:5) to obtain compound 10 (1.3631 g, 95% yield) as a white amorphous solid. 11H NMR (500 MHz, DMSO-d6) δ 8.01 (t, J = 5.4 Hz, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.72 (t, J = 5.5 Hz, 1H), 7.71 (t, J = 8.2 Hz, 1H), 7.62 (d, J = 8.3 Hz, 1H), 7.01 (d, J = 8.2 Hz, 1H), 6.73 (t, J = 5.3 Hz, 1H), 4.10 - 4.03 (m, 1H), 3.42 - 3.33 (m, 6H), 3.22 - 3.10 (m, 18H), 3.04 - 2.97 (m, 4H), 2.90 - 2.83 (m, 2H), 2.57 - 2.32 (m, 4H), 2.04 (t, J = 7.5 Hz, 2H), 1.80 - 1.70 (quin, J = 7.5 Hz, 2H), 1.57 (quin, J = 7.8 Hz, 16H), 1.41 - 1.16 (m, 37H), 0.93 (t, J = 7.4 Hz, 24H). HRMS (ESI-TOF): calcd for [C 35 H 68 B 12 IN5O8] 2- 471.7667: found 471.7667.

[0054] <Synthesis of Compound 11 from Compound 10>

Chem.

[0055] <Synthesis of Compound 12 from Compound 11> [ka] Under an argon atmosphere, compound 11 (1.277 g, 0.961 mmol) and pteroylazide (324.0 mg, 0.961 mmol) were dissolved in dimethyl sulfoxide (4.0 mL), and then 1,1,3,3-tetramethylguanidine (481 μL, 3.84 mmol) was added at room temperature. After stirring at room temperature for 4 hours, the mixture was diluted with dichloromethane / methanol, and the compound was precipitated by adding diethyl ether. The mixture was filtered, and the resulting yellow solid was washed with diethyl ether and dried under reduced pressure.

[0056] The resulting yellow solid was dissolved in dichloromethane (3.0 mL) and methanol (3.0 mL), and a methanol solution of tetramethylammonium chloride (1.053 g, 9.61 mmol) (3.0 mL) was added at room temperature. After stirring for 30 minutes, the mixture was filtered, and the resulting orange solid was washed with ethanol / methanol (1:1, 20 mL) and then dried under reduced pressure.

[0057] The resulting orange solid was dissolved in acetonitrile (6.0 mL) and distilled water (6.0 mL), and Amberlite® IR-120 (12.0 g) was added at room temperature. After stirring for 12 hours, the mixture was filtered, the organic solvent was removed under reduced pressure, and water was removed by freeze-drying to obtain compound 12 (609.6 mg, 3-step yield 56%) as an orange solid. 1 H NMR (500 MHz, DMSO-d6) δ 8.71 (s, 1H), 8.04-7.78 (m, 7H), 7.84 (s, 1H), 7.71 (d, J = 6.2 Hz, 2H), 7.60-7.55 (m, 4H), 7.00 (d, J = 8.2 Hz, 2H), 4.54 (s, 2H), 4.12-4.02 (m, 2H), 3.50 (t, J = 5.4 Hz, 1H), 3.46-3.30 (m, 4H), 3.23-3.09 (m, 4H), 3.09-2.93 (m, 4H), 2.33 (s, 2H), 2.04 (t, J = 7.4 HRMS (ESI-TOF): calcd for [C 44 H 70 B 12 IN 11 O8] 2- , 568.7840: found 568.7834.

[0058] [Example 2] Synthesis of BC-IP [ka] Compound 13 (Ishii, S.; Nakamura, HJ Organomet. Chem. 2018, 865, 178) was synthesized according to a known method.

[0059] <Synthesis of Compound 14 from Compound 13> Under an argon atmosphere, compound 4 (448.5 mg, 1.155 mmol) was dissolved in acetonitrile (5.0 mL), and then potassium carbonate (1.596 g, 11.55 mmol) and compound 13 (619.7 mg, 1.155 mmol) were added at room temperature. After stirring under heating and reflux for 8 hours, water was added to quench the reaction, and the mixture was extracted three times with dichloromethane. The organic layer was diluted, dried over magnesium sulfate, filtered, and the solvent was removed under reduced pressure.

[0060] The residue was dissolved in dichloromethane (0.8 mL) and ethanol (6.5 mL), and a methanol solution of tetramethylammonium chloride (1.458 g, 13.3 mmol) (6.5 mL) was added at room temperature. After stirring for 30 minutes, the mixture was filtered, and the resulting white solid was washed with ethanol / methanol (1:1, 12 mL) and dried under reduced pressure.

[0061] The resulting white solid was dissolved in acetonitrile (4.0 mL) and distilled water (4.0 mL), and Amberlite® IR-120 (7.0 g) was added at room temperature. After stirring for 12 hours, the mixture was filtered, the organic solvent was removed under reduced pressure, and water was removed by freeze-drying to obtain compound 14 (500.4 mg, 3-step yield 70%) as a white solid. 1H NMR (400 MHz, D2O) δ 7.65 (d, J = 8.0 Hz, 2H), 7.13 (d, J = 8.0 Hz, 2H), 3.83-3.62 (m, 8H), 3.29-3.23 (m, 2H), 3.16-3.11 (m, 2H), 2.66-2.61 (m, 2H), 2.31-2.24 (m, 2H), 1.96-1.92 (m, 2H), 1.76-1.73 (m, 2H), 1.54-1.49 (m, 2H), 1.44-1.35 (m, 4H).

[0062] [Example 3] Synthesis of the molecule PBC-IP-N3 for PET imaging [ka] Compound 15 (Robertson, M.; Bremner, JB; Coates, J.; Deadman, J.; Keller, PA; Pyne, SG; Somphol, K.; Rhodes, DI Eur. J. Med. Chem. 2011, 46, 4201) was synthesized according to known methods.

[0063] <Synthesis of Compound 16 from Compound 15> [ka] Under an argon atmosphere, compound 15 (1.041 g, 4.00 mmol) was dissolved in dichloromethane (12 mL), and then triethylamine (836 μL, 6.00 mmol) and bromoacetyl chloride (362 μL, 4.40 mmol) were added at 0°C. After stirring for 50 minutes, the reaction was stopped by adding saturated sodium bicarbonate aqueous solution, and the mixture was extracted three times with dichloromethane. The organic layer was washed with saturated brine, dried over magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The resulting crude product was purified by silica gel column chromatography (hexane / ethyl acetate = 55:45) to obtain compound 16 (1.258 g, yield 83%) as a yellow liquid. 1 H NMR (400 MHz, CDCl3) δ 6.99 (d, J = 1.9 Hz, 1H), 4.60-4.54 (m, 2H), 3.88 (s, 2H), 3.75 (s, 3H), 3.09 (q, J = 6.3 Hz, 2H), 1.93-1.84 (m, 1H), 1.78-1.68 (m, 1H), 1.53-1.43 (m, 13H).

[0064] <Synthesis of Compound 17 from Compound 16> [ka] Under an argon atmosphere, compound 16 (3.014 g, 7.91 mmol) was dissolved in N,N-dimethylformamide (15 mL), and then sodium azide ((1.541 g, 23.7 mmol)) was added at room temperature. After stirring at 50°C for 2 hours, water was added to quench the reaction, and the mixture was extracted three times with ethyl acetate. The organic layer was washed with 1 M hydrochloric acid, dried over magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The resulting crude product was purified by silica gel column chromatography (hexane / ethyl acetate = 55:45) to quantitatively obtain compound 17 (2.7477 g) as a colorless liquid. 1 H NMR (400 MHz, CDCl3) δ 6.81 (d, J = 6.8 Hz, 1H), δ 4.62-4.57 (m, 2H), 4.04-3.95 (m, 2H), 3.74 (s, 3H), 3.09 (q, J = 6.3 Hz, 2H), 1.91-1.82 (m, 1H), 1.76-1.67 (m, 1H), 1.52-1.42 (m, 11H), 1.37-1.30 (m, 2H).

[0065] <Synthesis of Compound 18 from Compound 17> [ka] Compound 17 (1.889 g, 5.50 mmol) was dissolved in methanol (17.6 mL) and distilled water (4.4 mL), and then lithium hydroxide monohydrate (1.154 g, 27.5 mmol) was added at room temperature. After stirring for 30 minutes, the reaction was stopped by adding 1 M HCl, and the mixture was extracted three times with dichloromethane. The organic layer was washed with saturated brine, dried over magnesium sulfate, filtered, and the solvent was removed under reduced pressure to obtain compound 18 (1.4758 g, yield 81%) as a yellow liquid. 1 H NMR (400 MHz, CDCl3) δ 11.4 (brs, 1H), δ 7.17 (d, J = 7.8 Hz, 1H), 4.56 (brs, 1H), 3.98 (s, 2H), 3.04 (brs, 2H), 1.92-1.83 (m, 1H), 1.77-1.68 (m, 1H), 1.47-1.35 (m, 13H).

[0066] <Synthesis of Compound 19 from Compound 18> [ka] Under an argon atmosphere, compound 18 (420.5 mg, 1.28 mmol), compound 9 (931.7 mg, 1.28 mmol), and HOBt·H2O (183.0 mg, 1.41 mmol) were dissolved in dichloromethane (7.7 mL). Then, diisopropylethylamine (267 μL, 1.53 mmol) and EDCI·HCl (269.5 mg, 1.41 mmol) were added at 0°C. After stirring at 0°C for 2 hours, the reaction was stopped by adding saturated sodium bicarbonate aqueous solution, and the mixture was extracted three times with dichloromethane. The organic layer was washed with saturated brine, dried over magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The resulting crude product was purified by silica gel column chromatography (dichloromethane / methanol = 98:2) to obtain compound 19 (869.7 mg, yield 65%) as a white amorphous solid. 1H NMR (400 MHz, CDCl3) δ 8.69 (brs, 1H), 7.41 (d, J = 9.1 Hz, 1H), 4.77 (brs, 1H), 4.46 (q, J = 7.4 Hz, 1H), 4.09 (d, J = 16.1 Hz, 1H), 4.01 (d, J = 16.1 Hz, 1H), 3.94-3.66 (m, 5H), 3.54-3.51 (m, 1H), 3.41-3.36 (m, 1H), 3.29-3.16 (m, 17H), 3.06 (brs, 2H), 1.95-1.86 (m, 4H), 1.67-1.59 (m, 16H), 1.49-1.39 (m, 27H), 0.99 (t, J = 7.3 Hz, 2H); 13 C NMR (125 MHz, CDCl3) δ 172.1, 167.8, 156.0, 78.5, 71.8, 69.9, 69.2, 58.9, 54.3, 51.9, 41.3, 40.3, 32., 29.3, 28.4, 24.1, 23.1, 19.7, 13.7.

[0067] <Synthesis of Compound 20 from Compound 19> [ka] Under an argon atmosphere, compound 19 (860.0 mg, 0.826 mmol) was dissolved in acetonitrile (12.0 mL), and then a 1,4-dioxane hydrochloride solution (4.0 M, 4.0 mL) was added at 0°C. After stirring at 0°C for 1 hour, the reaction was stopped by adding saturated sodium bicarbonate aqueous solution, and the mixture was extracted three times with dichloromethane. The organic layer was washed with saturated brine, dried over magnesium sulfate, filtered, and the solvent was removed under reduced pressure to obtain compound 20 (728.8 mg, yield 94%) as a white amorphous solid. HRMS (ESI-TOF): calcd for [C 12 H 34 B 12 N6O4] 2- 455.3766: found 455.3773.

[0068] <Synthesis of Compound 22 from Compound 20> [ka] Under an argon atmosphere, compound 20 (990.0 mg, 1.05 mmol), compound 8 (791.6 mg, 1.105 mmol), and HOBt·H2O (165.7 mg, 1.16 mmol) were dissolved in dichloromethane (6.3 mL). Then, diisopropylethylamine (220 μL, 1.26 mmol) and EDCI·HCl (221.8 mg, 1.16 mmol) were added at 0°C. After stirring at room temperature for 2 hours, the reaction was stopped by adding saturated sodium bicarbonate aqueous solution, and the mixture was extracted three times with dichloromethane. The organic layer was washed with saturated brine, dried over magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The resulting crude product was purified by silica gel column chromatography (dichloromethane / methanol = 98:2) to obtain compound 21 (616.9 mg, yield 36%) as a white amorphous solid. 13 C NMR (125 MHz, CDCl3) δ173.0, 172.8, 172.7, 172.1, 172.0, 167.8, 156.1, 141.7, 137.2, 130.7, 90.6, 78.7, 71.7, 69.7, 69.1, 58.7, 54., 53.9, 51.7, 41.3, 40.1, 39.0, 39.0, 38.8, 35.7, 34.7, 34.7, 34.7, 32.5, 31.9, 31.1, 29.5, 29.1, 28.8, 28.4, 28.2, 27.2, 27.1, 26.1, 26.0, 23.9, 23.0, 22.8, 19.6, 13.6; HRMS (ESI-TOF): calcd for [C 43 H 81 B 12 IN 10 O 10 ] 2- 577.3204: found 577.3209.

[0069] Under an argon atmosphere, compound 21 (316.5 mg, 0.193 mmol) was dissolved in acetonitrile (3.9 mL), and then a 1,4-dioxane hydrochloride solution (4 M, 0.97 mL) was added at 0°C. After stirring at 0°C for 1 hour, the reaction was stopped by adding saturated sodium bicarbonate aqueous solution, and the mixture was extracted five times with dichloromethane. The organic layer was washed with saturated brine, dried over magnesium sulfate, filtered, and the solvent was removed under reduced pressure to quantitatively obtain compound 22 (336.5 mg) as a white amorphous solid. HRMS (ESI-TOF): calcd for [C 38 H 73 B 12 IN 10 O8] 2- 527.2940: found 527.2937.

[0070] <Synthesis of Compound 24 from Compound 22> [ka] Under an argon atmosphere, compound 22 (152.8 mg, 0.105 mmol) and pteroylazide (33.7 mg, 0.100 mmol) were dissolved in dimethyl sulfoxide (1.0 mL), and then 1,1,3,3-tetramethylguanidine (50 μL, 0.400 mmol) was added at room temperature. After stirring at room temperature for 2 hours, the mixture was diluted with dichloromethane / methanol, and the target product was precipitated by adding diethyl ether. The mixture was filtered, and the resulting yellow solid was washed with acetone and diethyl ether, and then dried under reduced pressure to obtain compound 23 (96.5 mg, yield 61%) as a yellow solid. HRMS (ESI-TOF): calcd for [C 52 H 83 B 12 IN 16 O 10 ] 2- 674.3376: found 674.3372.

[0071] The resulting yellow solid (92.0 mg, 0.0582 mmol) was dissolved in dichloromethane (1.0 mL) and methanol (1.0 mL), and a methanol solution of tetramethylammonium chloride (110.9 mg, 0.582 mmol) (1.0 mL) was added at room temperature. After stirring for 30 minutes, the mixture was filtered, and the resulting orange solid was washed with ethanol / methanol (1:1, 20 mL), dried under reduced pressure, and compound 24 (63.0 mg, yield 73%) was obtained as a yellow solid. HRMS (ESI-TOF): calcd for [C 52 H 83 B 12 IN 16 O 10 ] 2- 674.3376: found 674.3381.

[0072] [Example 4] Synthesis of PET molecular platform BCN-F [ka] Compound 25 (DeForest, CA; Tirrell, DA Nat. Mater. 2015, 14, 523) was synthesized according to known methods.

[0073] <Synthesis of Compound 26 from Compound 25> [ka] Under an argon atmosphere, compound 25 (190.0 mg, 0.652 mmol) was dissolved in dichloromethane (3.3 mL), and then triethylamine (178 μL, 1.30 mmol) and triglycolamine (108 μL, 0.783 mmol) were added at 0°C. After stirring at 0°C for 1 hour, the reaction was stopped by adding saturated sodium bicarbonate aqueous solution, and the mixture was extracted three times with dichloromethane. The organic layer was washed with saturated brine, dried over magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The resulting crude product was purified by silica gel column chromatography (hexane / ethyl acetate = 40:60) to obtain compound 26 (207.1 mg, 98% yield) as a colorless liquid. 1 H NMR (400 MHz, CDCl3) δ 5.30 (brs, 1H), 4.16 (d, J = 8.1 Hz, 2H), 3.76 (t, J = 4.2 Hz, 2H), 3.66-3.52 (m, 9H), 3.39 (q, J = 5.1 Hz, 2H), 2.29-2.18 (m, 5H), 1.2-1.57 (m, 2H), 1.41-1.32 (m, 1H), 0.97-0.92 (m, 2H).

[0074] <Synthesis of Compound 27 from Compound 26> [ka] Under an argon atmosphere, compound 26 (98.1 mg, 0.302 mmol) was dissolved in dichloromethane (1.5 mL), and then triethylamine (84 μL, 0.603 mmol) and tosyl chloride (63.2 mg, 0.332 mmol) were added at 0°C. After stirring at 0°C for 3 hours, the reaction was stopped by adding saturated sodium bicarbonate aqueous solution, and the mixture was extracted three times with dichloromethane. The organic layer was washed with saturated brine, dried over magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The resulting crude product was purified by silica gel column chromatography (hexane / ethyl acetate = 50:50) to obtain compound 27 (31.2 mg, yield 22%) as a colorless liquid. 1 H NMR (400 MHz, CDCl3) δ 7.80 (d, J = 8.3 Hz, 2H), 7.34 (d, J = 8.3 Hz, 2H), 5.12 (brs, 1H), 4.19-4.11 (m, 4H), 3.71-3.68 (m, 2H), 3.60-3.51 (m, 6H), 3.35 (q, J = 5.2 Hz, 2H), 2.45 (s, 3H), 2.32-2.18 (m, 6H), 1.59-1.56 (m, 2H), 1.37-1.33 (m, 1H), 0.96-0.91 (m, 2H).

[0075] <Synthesis of Compound 28 from Compound 27> [ka] Under an argon atmosphere, compound 27 (10.7 mg, 0.0223 mmol) was dissolved in acetonitrile (1.5 mL), and then tetrabutylammonium fluoride solution (1.0 M, 89 μL) was added at room temperature. After stirring at 95°C for 10 hours, the reaction was stopped by adding saturated sodium bicarbonate aqueous solution, and the mixture was extracted three times with ethyl acetate. The organic layer was washed with saturated brine, dried over magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The resulting crude product was purified by silica gel column chromatography (hexane / ethyl acetate = 30:70) to obtain compound 28 (3.0 mg, yield 41%) as a colorless liquid. 1 H NMR (400 MHz, CDCl3) δ 5.15 (brs, 1H), 4.65-4.63 (m, 1H), 4.53-4.51 (m, 1H), 4.15 (d, J = 8.0 Hz, 2H), 3.80-3.76 (m, 1H), 3.72-3.63 (m, 5H), 3.57 (t, J = 5.3 Hz, 2H), 3.39 (q, J = 5.0 Hz, 2H), 2.33-2.17 (m, 6H), 1.63-1.57 (m, 2H), 1.42-1.32 (m, 1H), 0.97-0.92 (m, 2H).

[0076] [Example 5] Synthesis of PBC-IP PET Probe [ka] <Synthesis of compound 29 from compound 23 and compound 28> Under an argon atmosphere, compound 23 (3.8 mg, 0.00246 mmol) and compound 28 (0.8 mg) were dissolved in dimethyl sulfoxide (0.2 mL). After stirring at 40°C for 2 hours, the solvent was removed by freeze-drying to obtain compound 29 (4.5 mg) as a yellow solid. LRMS (ESI-TOF): calcd for [C 69 H 109 B 12 FIN 17 O 14 ] 2- 838.43: Found 838.41.

[0077] [Example 6] Evaluation of the binding of PBC-IP to human serum albumin The binding ability of PBC-IP to human serum albumin was evaluated using the following method. For comparison, the binding ability of BC-IP without folate and MID (International Publication WO2017 / 026276), which has been confirmed to covalently bind to albumin, was also evaluated in the same manner.

[0078] Human serum albumin (HSA, 10 μM, 200 μL) was mixed with boron compounds (MID, PBC-IP, BC-IP) (10 equivalents, 10 mM, 20 μL) in phosphate buffer and buffered saline (PBS), and reacted at 37°C for 1 hour. Next, this mixture was washed nine times (10,000 rpm, 4°C, 5 min) using a filter (Amicon R Ultra-0.5 mL, 30 K). Finally, the sample was diluted to a total of 5 mL with distilled water, and the boron concentration of the solution was measured using ICP-OES (iCAP 7400 Duo, Thermo), and affinity was calculated as the number of bound molecules per HSA molecule.

[0079] The results showed that PBC-IP binds 9.0 molecules to one albumin molecule. In contrast, MID binds 2.8 molecules and BC-IP binds 3.4 molecules, resulting in fewer binding molecules compared to PBC-IP.

[0080] [Example 7] Verification of folate receptor (FRα) expression levels in various human cancer cells (1)Western Blotting Analysis of FR Expression HeLa (human cervical cancer), MCF-7 (human breast cancer), U-87 MG (human brain tumor), CT26 (mouse colorectal cancer), A549 (human lung cancer) - 1 x 10 cells each 6 Cells were added to SDS-PAGE sample buffer (1 mL) and boiled at 95°C for 5 minutes. Cell lysates were then electrophoresed on a 10% SDS polyacrylamide gel and transferred to a PVDF membrane. Anti-FRα antibody (primary antibody) was added, and the cells were incubated at 4°C for 12 hours, followed by three washes with TBS buffer. HRP-conjugate secondary antibody was added, and the cells were incubated at room temperature for another hour. After adding the detection reagent, protein expression levels were detected by chemiluminescence intensity (Figure 1). As shown in Figure 1, folate receptors were relatively highly expressed in U87MG cells, HeLa cells, and CT26 cells, while expression was low in MCF-7 cells and A549 cells.

[0081] (2)Flow Cytometry Analysis of FR Expression HeLa, MCF-7, U-87 MG, CT26, or A549 cells (5 × 10⁻¹⁰) 5The cells were washed with 1 mL of PBS and immunoblocking solution was added. After incubation at 4°C for 15 minutes, the solution was centrifuged (3 minutes, 2000 rpm). Next, the supernatant was removed, primary antibody (20 μg / mL in PBS) was added, and after incubation at 4°C for 30 minutes, the solution was centrifuged and the supernatant was removed. After washing three times with PBS, FITC-labeled secondary antibody (2 μg / mL in PBS) was added. After incubation at 4°C for 30 minutes, the solution was centrifuged, the supernatant was removed, and washed three times with PBS. Then, this cell pellet was lysed in 500 μL of PBS. Fluorescence signals were observed using a flow cytometer (Figure 2). As shown in Figure 2, FRα expression was elevated in U87MG cells, HeLa cells, and CT26 cells.

[0082] [Example 8] Verification of the BNCT antitumor effect of PBC-IP (in vitro) Cells seeded in a 96-well plate (A549 1000 cells / well or U-87 MG 500 cells / well) were treated with 25 ppm [ 10 Cells were cultured at 37°C for 3 hours with PBC-IP and L-BPA-fructose at the concentration of [B], and then irradiated with thermal neutrons from one side of the plate for 12 minutes. Subsequently, the culture medium was changed and the cells were cultured for 96 hours, after which the MTT assay was performed to evaluate cell viability (Figure 3). Thermal neutron irradiation was performed using the irradiation facility at the Kyoto University Institute for Integrated Radiation Research (KUR), and the irradiated cells were cultured in the controlled area of ​​KUR. The viability was calculated based on the following formula.

number

[0083] As shown in Figure 3, PBC-IP showed a dose-dependently higher BNCT antitumor effect than BPA against U87MG cells, while BPA showed a higher BNCT antitumor effect against A549 cells. These results suggest that PBC-IP was taken up via FRα.

[0084] [Example 9] Verification of the pharmacokinetics of PBC-IP and the antitumor effect of BNCT (in vivo) Preparation of PBC-IP-HSA: HSA (170.3 mg, 2.56 μmol) and PBC-IP (59.8 mg, 50.9 μmol) were dissolved in PBS (1.8 mL) and stirred at 37 °C for 23 hours. This solution was subjected to 30 K ultrafiltration (Amicon Ultra-0.5 mL, Merck Millipore Ltd.), and the boron concentration was adjusted to 2500 ppm by ICP-OES measurement. The resulting PBS solution of the PBC-IP-albumin complex (PBC-IP-HSA) was used in the following experiments.

[0085] (A) Pharmacokinetics U-87MG cells were subcutaneously transplanted into the right thigh of nude mice (Balb / cSlc-nu / nu, female, 5-6 weeks old, 14-20 g). The mice were fed a normal solid diet and water and maintained in ambient atmosphere under a 12-hour light / dark cycle. When the tumor size reached 5-7 mm in diameter, 200 μL of PBS solution of boron compounds (PBC-IP-HSA and BPA: 25 mg B / kg) was injected into the tail vein of the mice. Three hours after injection (BPA) and six hours after injection (PBC-IP-HSA), the mice were lightly anesthetized, and blood samples were collected by cardiac puncture. Next, the mice were dislocated at the cervical vertebrae and dissected. The liver, kidneys, spleen, and tumors were excised, washed with 0.9% NaCl solution, and weighed. The excised organs were treated with 1 mL of concentrated nitric acid (ultra-trace analysis grade) at 90°C for 2 hours, and the decomposed samples were diluted with distilled water. After filtration with a hydrophobic filter, the boron concentration was measured by ICP-OES (Figure 4A). As shown in Figure 4A, PBC-IP-HSA showed greater accumulation in tumors than BPA. In addition, accumulation was observed in the liver and spleen, in addition to tumors.

[0086] (B) BNCT antitumor effect As described in (A), U-87MG tumor-bearing mice were given a 200 μL PBS solution of boron compounds (PBC-IP-HSA, L-BPA: 25 mg each). 10B) / kg) was injected via the tail vein. The entire body of the mouse was placed in an acrylic mouse holder and fixed to a 5mm thick thermoplastic plate. Three hours (L-BPA) or six hours (PBC-IP-HSA) after administration, 4.5-6.1 × 10¹⁴ units were injected into the right thigh of the mouse in the KUR reactor. 12 neutrons / cm 2 Neutrons were irradiated within the dose range. The BNCT effect was evaluated based on the change in tumor volume in mice (Figure 4B). To determine the tumor volume, the two vertical diameters of the tumor were measured with a slide caliper, and the spherical volume formula {4 / 3π × (R / 2) 3 The calculation was performed using}, where R is the average of the longest and shortest dimensions of the tumor (in millimeters, respectively). As shown in Figure 4B, PBC-IP-HSA(25 mg( 10 B) / kg, 6h after) L-BPA (25 mg ( 10 A significantly higher BNCT antitumor effect was obtained compared to B) / kg, 3h after.

[0087] (C) Weight change after BNCT irradiation In the irradiation experiment conducted in (B), changes in body weight after irradiation were measured. As shown in Figure 4C, no significant difference in body weight change was observed in either the BPA irradiation group or the PBC-IP-HSA irradiation group compared to the control group, indicating that PBC-IP-HSA has sufficiently low toxicity.

[0088] [Example 10] Selective accumulation ability of PBC-IP in various cells Regarding the cellular accumulation of the PBC-IP complex, a comparison was made with existing boron-containing drugs (BPA) using human lung cancer cells (A549), human glioblastoma cells (U87MG), and rat glioblastoma cells (C6, F98). The results showed that PBC-IP was 20 to 150 times superior to BPA in terms of cellular accumulation and intracellular retention (Figure 5). Of these four cell types, BPA showed selective accumulation in A549 cells, which highly express LAT1, while PBC-IP showed selective accumulation in U87MG and F98 cells.

[0089] [Experimental Method] (cell) F98 rat glioblastoma cells were provided by Dr. Rolf Barth (Department of Pathology, The Ohio State University, Columbus, OH, US) and cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 10% penicillin / streptomycin / amphotericin B at 5% CO2 and 37°C. The F98 rat glioblastoma cells were histologically characterized as anaplastic astrocytomas. All cell culture materials were purchased from Gibco Invitrogen Corporation (Grand Island, NY).

[0090] (Intracellular boron accumulation concentration) First, 5 x 10 5 Each cell was placed in a 100 mm dish (Becton Dickinson, Franklin Lakes, New Jersey) and cultured in the above medium at 5% CO2 and 37°C. After 72 hours of culture, the medium was replaced with a medium containing 5 μg B / mL of BPA, BSH, or PBC-IP, and cultured for 2.5 hours, 6 hours, and 24 hours. Subsequently, the medium containing boron compounds was removed, the cells were washed twice with 4% phosphate-buffered saline (PBS), and then detached with trypsin-ethylenediaminetetraacetic acid solution. Then, the medium was added, and centrifugation (200 × g, 5 minutes) was performed twice, the cells were counted, and finally, sedimentation was performed. After that, the cells were digested overnight in 1 N nitrate solution (Wako Pure Chemical Industries, Osaka, Japan), and the amount of boron uptake was measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES) using an iCAP6300 emission spectrometer (Hitachi High-Technologies, Tokyo, Japan).

[0091] [Example 11] BNCT therapeutic effect of PBC-IP on F98 and C6 glioblastoma cell transplanted rat brain tumor models The optimal administration conditions for intravenous administration were investigated in the F98 rat model of malignant glioblastoma. With high-concentration, high-dose administration of PBC-IP, blood boron concentrations were found to be high (over 150 ppm, compared to approximately 20-30 ppm for BPA). To ensure safety, it was determined that an effective treatment could be expected with approximately 1 / 10th of the dose. Furthermore, dose-increase studies were conducted in the low-dose range to determine the optimal dose, revealing that the toxicity of this drug tends to depend on the concentration, not the dose. Local administration using Convection Enhanced Delivery (CED) showed consistently high intratumoral boron accumulation of approximately 25 ppm. The accumulation ratio compared to normal brain tissue was over 30 times, while the blood concentration was extremely low, resulting in a concentration ratio of approximately 100 times.

[0092] Using F98 and C6 transplanted rat brain tumor models, the optimal administration conditions for local administration (CED) of PBC-IP were investigated (Figure 6). With intravenous administration of BPA (10 mg B / kg), the F98 tumor boron concentration was approximately 16 ppm one hour after administration, with a normal brain tissue (T / N) ratio of 5.4 and a blood concentration ratio (T / B) of 2.2. On the other hand, with low-dose PBC-IP administration (0.5 mg B / kg), the F98 tumor boron concentration was approximately 26.5 ppm three hours after PBC-IP (CED) administration, with an extremely high normal brain tissue (T / N) ratio of 29 and a blood concentration ratio (T / B) of 92. Similarly, the C6 tumor boron concentration reached 36.6 ppm, with a normal brain tissue (T / N) ratio of 63 and a blood concentration ratio (T / B) of 78, both of which were high.

[0093] Three hours after administration of PBC-IP (CED), neutron irradiation was performed to verify the therapeutic effect of neutron capture therapy (Figure 7). The brain tumor model was divided into groups: untreated, neutron irradiation only, BNCT with BPA (iv), BNCT with local administration of PBC-IP (CED), and a combination group of PBC-IP (CED) with BPA (iv). Treatment experiments were conducted, and survival time after tumor transplantation was compared. The treatment effect of intravenous BPA, used as a control for BNCT treatment, showed a significant extension of survival time, with a median survival time of 37 days. In contrast, the survival extension effect of the PBC-IP (CED) group was extremely high, with more than half still alive at the 90-day observation point, and the median survival time had not yet been calculated, indicating excellent results. The group that combined intravenous BPA with PBC-IP (CED) monotherapy showed an even greater effect, demonstrating the antitumor effect of PBC-IP on BPA-refractory tumor areas. In the C6 transplanted rat brain tumor model, the tumor boron concentration induced by PBC-IP (CED) was approximately 36 ppm, which is higher than that of F98. Although neutron irradiation experiments in this model have been limited to a few pilot studies, long-term survival in rats was achieved only with PBC-IP (CED).

[0094] We performed neutron irradiation after local administration (CED) of the PBC-IP complex into the rat brain, and pathologically examined the effects of BNCT using the same drug on normal brain tissue. No obvious physical or neurological effects were observed during the observation period after irradiation, and pathological examination of the brain tissue after neutron irradiation revealed no obvious tissue adverse events other than mild tissue reactions limited to the catheter insertion site and surrounding areas used for local administration (CED) (Figure 8).

[0095] [Experimental Method] (F98 brain tumor orthotopic model rat) All male Fisher rats weighing 200-240g (F344 Japan SLC; Hamamatsu, Shizuoka, Japan) were anesthetized by intraperitoneal injection of a mixture of three anesthetics: medetomidine (0.4 mg / kg), midazolam (2.0 mg / kg), and butorphanol (5.0 mg / kg). They were then fixed to a stereotactic frame (Model 900; David Kopf Instruments, Tujunga, California). Next, a midline incision was made in the scalp, and 1mm burr holes were drilled using an electric drill, 1mm posterior and 4mm to the right of the bregma. A 25μL Hamilton syringe (model 1700RN; Hamilton Bonaduz, Switzerland) with a 26-gauge needle was inserted into the rat brain, and F98 tumor cells were transplanted. The needle was first inserted to a depth of 6mm from the dura mater, and then withdrawn to a distance of 1mm from the target in the brain (5mm from the dura mater). In the therapeutic experiment, the F98 cell suspension diluted with 10 μL of DMEM containing 1.4% agarose was used. 3 In in vivo distribution experiments, 10 5 The solution was injected at a rate of 20 μL / min using an automatic injection pump to achieve the desired concentration. Immediately after injection, the needle was withdrawn, the burr hole was covered with bone wax, and the scalp was sutured.

[0096] (CED method) CED is a direct drug administration method that allows for localized drug delivery into the interstitial tissue of the brain under sustained low positive pressure, achieving high concentrations and broad distribution of the drug (Yin et al. Cancer Gene Ther. 2013, 20: 336-341; Bobo et al. Proc. Natl. Acad. Sci. USA, 1994, 91: 2076-2080). For drug CED, an Alzet osmotic pump (model #2001D; DURECT Corporation, Cupertino, California) and an intracerebral infusion kit (rigid stainless-steel cannula, 5-mm 28 gauge) were assembled and filled with 200 μL of boron compound solution. After anesthetizing rats carrying F98 glioblastoma cells, the infusion pump was subcutaneously implanted in the rats' backs. A needle connected to the infusion cannula was inserted through the same burr hole into which the tumor cells had been transplanted. This infusion pump was able to administer boron compounds at a rate of 8 μL / h for more than 24 hours.

[0097] (Boron concentration in each tissue) Fourteen days after tumor transplantation, rats carrying F98 malignant glioblastoma cells were administered each boron compound (BPA and PBC-IP). The in vivo distribution of BPA was measured at 2 and 6 hours after intravenous administration of 12 mg B / kg body mass (bm), and at 2, 6, and 24 hours after the completion of PBC-IP (CED) administration, using 3 to 5 rats per group. Rats were euthanized after each administration, and tumors, normal brains, blood, hearts, lungs, livers, spleens, kidneys, skin, and muscles were excised, weighed, and digested with 1 N nitrate solution. The boron concentration (μg B / g) in each organ was measured by ICP-AES.

[0098] (Neutron irradiation experiment) In the BNCT experiment, 10 3Fourteen days after transplantation of individual F98 glioblastoma cells, neutron irradiation was performed using KURRI. Glioblastoma cell-carrying rats were randomly divided into five groups (Groups 1-5), with each group consisting of 6-10 rats. Group 1 was the untreated control group; they were transported to the reactor, anesthetized, and treated the same as the other groups except for neutron irradiation (sham irradiation). Group 2 was the neutron irradiation control group. Group 3 was the group that received intravenous BPA followed by neutron irradiation. Group 4 was the group that underwent PBC-IP using CED. 10 This group received neutron irradiation after administration of B-enrich. Group 5 was PBC-IP by CED. 10 This group received a combination of B-enrich (B-enrich) and intravenous BPA injection followed by neutron irradiation (combination group). After anesthetizing the rats with a mixed anesthetic, their entire bodies except the head were shielded and they were fixed to a board. Two hours after the end of intravenous administration or three hours after the end of CED, they were irradiated with neutrons from a 5 MW reactor for 20 minutes. After neutron irradiation, the unirradiated and neutron-irradiated animals were left at KURRI for observation. The therapeutic effect was evaluated by the survival time of all rats.

[0099] All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety. [Industrial applicability]

[0100] Since the boron-containing drug of the present invention is used as a pharmaceutical, the present invention is applicable to industries related to pharmaceuticals.

Claims

1. The following equation (II) 【Chemistry 1】 [In the formula, C represents a carbon atom, L2, L3, and L4 each independently represent divalent groups that function as spacers, X represents a group that binds to albumin, and Y represents, 10 B represents a group containing B, and Z is given by the following formula (D) 【Chemistry 2】 (In the formula, * represents a bonding site.) A boron agent for boron neutron capture therapy, characterized by containing a compound represented by ], which represents the group shown by .

2. In formula (II), L2, L3, and L4 are alkylene groups (wherein one or more of the alkylene groups are -CH 2 The boron agent for boron neutron capture therapy according to claim 1, characterized in that - may be substituted with -O-, -S-, -NH-, or -CO-.

3. X in formula (II) is one of the following formulas (A) to (C) 【Transformation 3】 (In the formula, * represents a bonding site, and R represents a hydrogen atom, fluorine atom, chlorine atom, bromine atom, or iodine atom.) A boron agent for boron neutron capture therapy according to claim 1 or 2, characterized in that it is a group represented by .

4. The boron agent for boron neutron capture therapy according to any one of claims 1 to 3, characterized in that Y in formula (II) is a group derived from a boron cluster.

5. A compound represented by formula (II) is the following formula (IIa) 【Chemistry 4】 The boron agent for boron neutron capture therapy according to claim 1, characterized in that it is a compound represented by .

6. The following equation (IV) 【Transformation 5】 [In the formula, C represents carbon atom, 18 F represents a radioactive fluorine atom with mass number 18, L6, L7, L8, L9, and L10 each independently represent divalent groups that function as spacers, X represents a group that binds to albumin, and Y is 10 B represents a group containing B, and Z is given by the following formula (D) 【Transformation 6】 (In the formula, * represents a bonding site.) A PET imaging agent characterized by containing a compound represented by [], which represents the group shown by [].

7. In formula (IV), L6, L8, L9 and L10 are alkylene groups (wherein one or more of the alkylene groups are -CH 2 - may be substituted with -O-, -S-, -NH-, or -CO-. ) and L 7 However, alkylene group (wherein one or more alkylene groups are -CH 2 - may be substituted with -O-, -S-, -NH-, or -CO-, and one of the alkylene groups is -CH 2 - may be substituted with a divalent group formed by a click reaction.) The PET imaging agent according to claim 6.

8. X in formula (IV) is one of the following formulas (A) to (C) 【Transformation 7】 (In the formula, * represents a bonding site, and R represents a hydrogen atom, fluorine atom, chlorine atom, bromine atom, or iodine atom.) The PET imaging agent according to claim 6 or 7, characterized in that it is a group represented by .

9. The PET imaging agent according to any one of claims 6 to 8, characterized in that Y in formula (IV) is a group derived from a boron cluster.

10. The compound represented by formula (IV) is given by the following formula (IVa) 【Transformation 8】 The PET imaging agent according to claim 6, characterized in that it is a compound represented by

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