JP2023009974A - Compound comprising boron and iodine or salt thereof, and their use - Google Patents

Abstract

To provide: in one aspect, a boron-containing compound, which can be used in a boron neutron capture therapy and an accumulation amount of which in a tumor can be easily measured; and in another aspect, a boron-containing compound, a small dosage of which provides a high 10B-boron concentration in a tumor tissue.MEANS FOR SOLVING THE PROBLEM: Provided is: a compound comprising boron and iodine, represented by the following formula (I) (in formula (I), X, L, and Y respectively represent a group containing boron 10 (10B), a linker, and a group containing iodine); or a salt thereof.SELECTED DRAWING: None

Description

TECHNICAL FIELD The present invention relates to compounds or salts thereof containing boron and iodine, and more particularly to compounds or salts thereof used for boron neutron capture therapy.

Boron Neutron Capturing Therapy (hereinafter sometimes referred to as "BNCT") is a boron isotope, boron 10 ( ¹⁰ B), is administered to a patient suffering from a tumor disease, and then a neutron beam is administered. In recent years, this treatment method has attracted attention as a minimally invasive cancer treatment method because it is a treatment method in which only tumor cells are killed by irradiation, and the QOL after treatment is also excellent. Boron-10 ( ¹⁰ B) absorbs low-energy thermal neutrons and emits α-rays (helium nuclei) and ⁷ Li nuclei through a nuclear fission reaction ( ¹⁰ B(n, α) ⁷ Li reaction). Thermal neutrons have low energy (0.025 MeV) and are less harmful to the human body, while α rays and ⁷ Li nuclei produced by the nuclear fission reaction have sufficient energy (2.4 MeV) to destroy cells. have. In addition, since the range of α-rays emitted by the nuclear fission reaction is about 9 μm, and the range of ⁷ Li nuclei is about 4 μm, which is short, ¹⁰ B is preliminarily accumulated in tumor tissue, and thermal neutrons are irradiated thereon. For example, high-energy radiation is generated only in the vicinity of tumor tissue, and only tumor cells can be selectively destroyed. Boron neutron capture therapy is known to be particularly effective in treating head and neck cancers, such as highly malignant brain tumors in which cancer cells are mixed with normal cells, and melanoma.

In boron neutron capture therapy, boron-containing compounds are used to introduce boron-10 into tumor tissue. From the viewpoint of sufficiently destroying tumor cells and minimizing the effect on normal tissues, the boron-containing compound is preferably one that selectively and abundantly accumulates in tumor tissue, and the tumor tissue to be treated. It is said that it is desirable to set the ¹⁰ B concentration in 20 ppm or more (equivalent to 20 μg/g). This concentration is equivalent to about 2 mM when converted to molar concentration with the molecular weight of boron being about 10. This is an extremely high concentration compared to

A boron-containing compound amenable to use in boron neutron capture therapy, including clinical studies, is L-BPA (compound 1a, 4-borono-L-phenylalanine ( ¹⁰ B), CAS RN: 80994-59-8), part ¹⁸ F-labeled [ ¹⁸ F]F-BPA (compound 1b, ¹⁸ F-fluoro-borono-L-phenylalanine), boric acid, BSH (compound 2, sodium mercaptododecaborate ( ¹⁰ B), CAS RN: 12448-24- 7). In addition, L-BPA (compound 1a) was marketed in Japan in 2020 as a drug for BNCT (Non-Patent Document 1).

L-BPA (compound 1a) has been used as a drug for BNCT based on its safety. equivalent), a large amount of L-BPA is administered by infusion. On the other hand, BSH (Compound 2) is a divalent anion cluster with 12 boron atoms per molecule. extremely low. As described above, existing boron-containing compounds used for BNCT, that is, BNCT agents, all have room for improvement in their accumulation in tumors. Development of boron-containing compounds that are selectively and extensively incorporated into tissues is highly desired.

Since BSH has up to 12 ¹⁰ B atoms per molecule, if its accumulation in tumors can be enhanced, a dramatic increase in intracellular boron concentration is expected. From this point of view, many techniques for increasing the accumulation of BSH in tumors have been reported. However, according to Non-Patent Document 2, even when a cell-permeable peptide is used, a high concentration of 1 mM or more is still required to give an intracellular boron concentration of 20 ppm (corresponding to approximately 2000 ng/10 ⁶ cells). of BSH is required.

On the other hand, in boron neutron capture therapy, it is desirable to generate a sufficient dose of radiation to destroy tumor cells in the tumor tissue being treated, while minimizing the effect on normal tissue. In addition to boron, other elements in living organisms, especially hydrogen and nitrogen, which are abundant in living organisms, absorb neutrons and emit radiation. However, excessive neutron beam irradiation is not preferable, and it is extremely important to irradiate a necessary and sufficient amount of neutron beams based on the boron concentration in the tumor. However, there is no established method for measuring the boron concentration in the tumor at the present stage. For example, as shown in Non-Patent Documents 1 and 3, instead of directly measuring the boron concentration in the tumor , and the current situation is that the whole blood boron concentration is used as an alternative index.

As a method for measuring the amount of boron accumulated in a tumor, the use of positron emission tomography (Positron Emission Tomography, hereinafter sometimes simply referred to as "PET") has been attempted (Non-Patent Document 4). ¹⁸ F-FBPA ( ¹⁸ F-fluoro-borono-L-phenylalanine) is known as a boron-containing compound that can be imaged at . However, the measurement of the amount of accumulated boron by PET imaging requires time from diagnosis to treatment, which imposes a heavy burden on the patient. In addition, PET imaging requires the handling of radioactive agents (eg, ¹⁸ F), which inevitably exposes medical personnel to radiation.

Patent Publication No. 6548060

Steboronine Intravenous Infusion Bag 9000mg/300mL, Pharmaceutical Interview Form, Revised May 2020 (2nd Edition) Michiue H, et al. Journal of Controlled Release, 330, 788-796 (2021) Ono K, et al. Advance in Neutron Capture Therapy 2006, 27-30 Yoshihiro Takai, "Boron Neutron Capture Therapy and Molecular Imaging -The Meaning and Expectations of 18F-FBPA-PET-", RADIOISOTOPES, 68, 25-35 (2019)

The present invention has been made in view of the above-described problems of the prior art, and in one aspect, provides a boron-containing compound that can be used for boron neutron capture therapy and that can easily measure the amount accumulated in tumors. The task is to provide Another object of the present invention is to provide a boron-containing compound that provides a high ¹⁰ B-boron concentration in tumor tissue when administered in small doses.

In the process of earnest research efforts to solve the above problems, the present inventors have found that, as a medical agent used at a concentration similar to that of boron-containing compounds used in boron neutron capture therapy, it can be used for X-ray imaging. I focused on the fact that there is an iodine drug that can be used. Then, if a compound having both iodine with X-ray imaging ability and ¹⁰ B used for boron neutron capture therapy is created in one molecule, the iodine concentration, which is easier to measure than the boron concentration, can be measured and measured. By converting the iodine concentration obtained into the boron concentration, the boron concentration in any part of the living body can be easily and quickly grasped, and in addition to boron, by using a compound having iodine with excellent fat solubility, The present inventors have found that the permeation of the compound to the cell membrane is enhanced and that the intracellular boron concentration can be dramatically increased, and thus the present invention has been completed.

That is, the present invention solves the above problems by providing the following [1] to [9].

[1] A compound containing boron and iodine or a salt thereof, characterized by being represented by the following formula (I). In the formula (I) below, X represents a group containing boron 10 ( ¹⁰ B), L represents a linker, and Y represents an iodine-containing group.

[2] The compound or salt thereof according to [1] above, wherein X in the above formula (I) is a group containing a boron cluster containing boron 10 ( ¹⁰ B).

[3] The compound or salt thereof according to [1] or [2] above, wherein X in the above formula (I) is a group represented by the following formula (XI). In the formula (XI) below, a black circle represents BH, a white circle represents B, and * represents a bond with an adjacent atom.

[4] The compound or salt thereof according to any one of the above [1] to [3], wherein Y in the above formula (I) is a group represented by the following formula (YI). In the following formula (YI), R ¹ represents a halogen atom or an optionally substituted monovalent organic group, a represents an integer of 1 to 5, and when there are multiple R ¹ Each is independent, and * represents a bond with an adjacent atom. However, the group represented by formula (YI) below contains at least one or more iodine atoms.

[5] The compound or salt thereof according to any one of [1] to [4], wherein Y in formula (I) is a group represented by formula (YII) below. In the following formula (YII), ^R2 represents an iodine atom ^, R3 represents an optionally substituted monovalent alkyl group or an optionally substituted monovalent halogenated alkyl group, and b represents an integer of 1 to 4, and * represents a bond with an adjacent atom.

[6] The compound or salt thereof according to any one of [1] to [5], wherein Y in formula (I) is a group represented by formula (YIII) below. In formula (YIII) below, R ³ represents an optionally substituted monovalent alkyl group or an optionally substituted monovalent halogenated alkyl group, and * represents a bond with an adjacent atom. represent a hand.

[7] The compound or salt thereof according to any one of the above [1] to [6], wherein Y in the above formula (I) is a group represented by the following formula (YIV). In the formula (YIV) below, * represents a bond with an adjacent atom.

[8] The compound or salt thereof according to any one of the above [1] to [7], wherein L in the above formula (I) is a group represented by the following formula (LI). In addition, in the following formula (LI), * represents a bond with an adjacent atom.

The compound or salt thereof according to any one of [1] to [8] above contains boron ¹⁰ B in its basic skeleton, and thus can be suitably used as a boron drug in boron neutron capture therapy. That is, according to the present invention, the following [9] to [11] are further provided.

[9] A pharmaceutical composition comprising the compound or salt thereof according to any one of [1] to [8] above.

[10] The pharmaceutical composition according to [9] above, which is used for boron neutron capture therapy.

[11] A method of treating a tumor disease, comprising the steps of administering the compound or salt thereof according to any one of [1] to [8] above to a patient, and irradiating the patient with neutron beams.

Further, when a compound containing both boron and iodine or a salt thereof is used as a drug for BNCT, the boron concentration in any site in the body, such as a tumor, can be measured more easily than the boron concentration. can be obtained easily and quickly by measuring and converting the measured iodine concentration to the boron concentration. That is, according to the present invention, the following [12] is further provided.

[12], the step of administering the compound or salt thereof according to any one of the above [1] to [8] to a patient, the step of measuring the iodine concentration in the patient's tumor, based on the measured iodine concentration A method of treating a tumor disease, comprising the steps of estimating a boron concentration in a tumor, and irradiating the patient with neutron beams based on the estimated boron concentration.

According to the compound or its salt according to the present invention, and the pharmaceutical composition containing them, the boron concentration in vivo can be obtained simply and quickly by measuring the iodine concentration, which is easier to measure than the boron concentration. can.

FIG. 3 shows a synthetic scheme of compounds 3a-3c (BS-DIP-OMe, BS-DIP-OEt, BS-DIP-OEF). FIG. 3 shows the intracellular boron concentration in each cell after incubating mouse melanoma cells B16/BL6 with compounds 3a-3c (BS-DIP-OMe, BS-DIP-OEt, BS-DIP-OEF).

The present invention will be described in more detail below.

The compound or salt thereof according to the present invention is, as described above, a compound or a salt thereof characterized by being represented by the following formula (I).

In formula (I) above, X represents a group containing boron 10 ( ¹⁰ B), L represents a linker, and Y represents an iodine-containing group. Boron-10 is an isotope of boron with a mass number of ¹⁰ and is represented as 10B. There are two stable isotopes of boron 10 ( ¹⁰ B) and boron 11 ( ¹¹ B) in nature. occupy Boron neutron capture therapy uses the nuclear fission reaction caused by ¹⁰ B among these boron isotopes.

X in the above formula (I) may be basically any group as long as it is a group containing ¹⁰ B. For example, one It may be a group containing a boron atom, preferably a group containing 2 or more boron atoms, more preferably a group containing 3 or more boron atoms, and 6 or more boron atoms. A group containing a boron cluster is more preferable, and a group containing a boron cluster is particularly preferable.

As used herein, the term "boron cluster" means a molecule having a polyhedral structure formed mainly by aggregation of a plurality of boron atoms, preferably a boron cluster containing 3 to 20 boron atoms, and more Boron clusters containing 10 to 20 boron atoms are preferred, and boron clusters containing 10 to 12 boron atoms are more preferred. Boron clusters include, for example, Decaborane (B ₁₀ H ₁₄ ), Decahydrodecaborate ([B ₁₀ H ₁₀ ] ^2- ), Dodecaborate ([B ₁₂ H ₁₂ ] ^2- ), nido, nido-Octadecaborane (22) (B ₁₈ H ₂₂ , CAS RN: 21107-56-2), but not limited to borohydride compounds.

For example, the boron cluster may contain a carbon atom, a nitrogen atom, an oxygen atom, a sulfur atom, etc. in addition to the boron atom as a skeletal atom constituting the boron cluster. The number of atoms other than boron atoms contained in the boron cluster is preferably 0 to 5, more preferably 0 to 2. Boron clusters containing atoms other than boron atoms in the basic skeleton include, for example, carborane containing boron and carbon atoms. Examples of carborane include closo-carborane ([CB ₁₁ H ₁₂ ] ⁻ ) containing one carbon atom, closo-carborane (C ₂ B ₁₀ H ₁₂ ) containing two carbon atoms, and nido-carborane ([C ₂ B ₉ H ₁₁ ] ⁻ ), and, for example, sandwich-type compounds represented by the molecular formula M(C ₂ B ₉ H ₁₀ ) ₂ are known, but are not limited to these. In the molecular formula, M represents a transition metal element, such as Fe, Ni, Co, Mo, and the like. That is, the boron clusters may be metal complexes with transition metals such as Fe, Ni, Co, Mo.

The boron cluster used in the present invention may basically be any boron cluster, but from the viewpoint of increasing the water solubility of the compound or salt thereof according to the present invention, an ionic boron cluster Alternatively, it is preferably a water-soluble boron cluster. Examples of ionic or water-soluble boron clusters include dodecaborate ([B ₁₂ H ₁₂ ] ²⁻ ), dodecaborate having a thiol group ([B ₁₂ H ₁₁ SH] ²⁻ , BSH), dodecaborate having a hydroxyl group ([B ₁₂ H ₁₁ OH] ²⁻ ), dodecaborate having an amino group ([B ₁₂ H ₁₁ NH ₃ ] ⁻ ), and the like are known.

In one preferred embodiment, X in formula (I) above is a group containing a boron cluster, more preferably a monovalent group comprising a boron cluster. A monovalent group consisting of a boron cluster means a group excluding any one H constituting a boron cluster. For example, when the boron cluster is the aforementioned dodecaborate ([B ₁₂ H ₁₂ ] ^2- ), the monovalent group comprising the boron cluster is a group represented by formula (XI) below. In the formula (XI) below, a black circle represents BH, a white circle represents B, and * represents a bond with an adjacent atom. That is, the monovalent group consisting of boron clusters is covalently attached to the linker L via *.

The compound or its salt which concerns on this invention is used for boron neutron capture therapy in one suitable aspect|mode. Therefore, the isotope ratio of ¹⁰ B in X in the above formula (I) is preferably 19% or more, more preferably 50% or more, further preferably 70% or more, and 90% The above is even more preferable. The ¹⁰ B isotope ratio of the boron-containing compound can be increased by using boron with an increased ¹⁰ B content ( ¹⁰ B-enriched boron) as a raw material. As a method for producing ¹⁰ B-enriched boron, for example, a chemical exchange distillation method is known.

On the other hand, Y in the above formula (I) is a group containing an iodine atom. Y may be basically any group as long as it contains at least one or more iodine atoms, but the imaging ability of the compound or salt thereof according to the present invention in X-ray CT or fluorescent X-ray CT and / or from the viewpoint of increasing the lipophilicity of the compound or salt thereof according to the present invention and improving the permeability of the cell membrane, it is preferably a group containing two or more iodine atoms.

Further, from the viewpoint of increasing the lipid solubility of the compound or salt thereof according to the present invention and improving the permeability of the cell membrane, Y in the above formula (I) is a group having at least one or more phenyl rings. Preferably, for example, in a preferred embodiment, Y in formula (I) above may be a group represented by formula (YI) below.

In formula (YI) above, R ¹ represents a halogen atom or an optionally substituted monovalent organic group. a represents an integer of 1 to 5; When there is more than one R ¹ , each of them is independent. That is, all ^R1 's may be the same or different. However, the structure represented by the above formula (YI) contains at least one or more iodine atoms. Note that * represents a bond with an adjacent atom.

In R ¹ , the monovalent organic group is, for example, a monovalent group having 1 to 10 carbon atoms. The monovalent organic group may basically be any group, but examples include alkyl groups, alkoxy groups, phenyl groups, phenoxy groups, amino groups, imino groups, thioalkyl groups, allyl groups, and the like. can be done. These groups may be substituted with a substituent.

As used herein, the term "optionally substituted" includes both cases where substitutable hydrogen atoms constituting the group are unsubstituted and substituted. The case where substitutable hydrogen atoms are not substituted means that all substitutable hydrogen atoms are hydrogen atoms. On the other hand, the case where the substitutable hydrogen atom is substituted means that the substitutable hydrogen atom is substituted with a substituent other than a hydrogen atom. Examples of substituents include halogens, hydroxy groups, alkyl groups, alkenyl groups, alkynyl groups, acetyl groups, cycloalkyl groups, halogenated alkyl groups, haloalkyl groups, hydroxyalkyl groups, alkoxy groups, haloalkoxy groups, amino groups, nitro groups, cyano groups, carboxyl groups, and the like, but are not limited to these. A plurality of substituents may be used. When there are multiple substituents, those substituents may be the same or different.

In a preferred embodiment, Y in formula (I) above is a group represented by formula (YII) below.

In formula (YII) above, ^R2 represents an iodine atom ^, and R3 represents an optionally substituted monovalent alkyl group or an optionally substituted monovalent halogenated alkyl group. b represents an integer of 1 to 4; * represents a bond with an adjacent atom.

In R ² , the monovalent alkyl group is, for example, a monovalent alkyl group having 1 to 10 carbon atoms, more preferably 1 to 8 carbon atoms, more preferably 1 to 5 carbon atoms, and more More preferably, it is a monovalent alkyl group having 1 to 2 carbon atoms.

On the other hand, in R ³ , the monovalent halogenated alkyl group is, for example, a monovalent halogenated alkyl group having 1 to 10 carbon atoms, more preferably 1 to 8 carbon atoms, still more preferably carbon atoms It is a monovalent halogenated alkyl group having 1 to 5 carbon atoms, more preferably 1 to 2 carbon atoms. Alkyl groups and halogenated alkyl groups may be linear or branched. Here, a halogenated alkyl group refers to a group in which one or more hydrogen atoms contained in an alkyl group are substituted with halogen atoms. Halogen atoms include fluorine, chlorine, bromine, iodine, and antimony atoms. Examples of the halogenated alkyl group in which one hydrogen atom in the alkyl group having 1 to 10 carbon atoms is substituted with a halogen atom include fluoromethyl group, 2-fluoroethyl group, 3-fluoropropyl group, chloro Examples include, but are not limited to, methyl group, 2-chloroethyl group, 3-chloropropyl group, bromomethyl group, 2-bromomethyl group, 3-bromopropyl group. On the other hand, the halogenated alkyl group in which two or more hydrogen atoms in the alkyl group having 1 to 10 carbon atoms are substituted with halogen atoms includes a difluoromethyl group, a trifluoromethyl group, a tetrafluoroethyl group and a pentafluoroethyl group. groups, dichloromethyl groups, trichloromethyl groups, tetrachloroethyl groups, pentachloroethyl groups, and the like, but are not limited to these.

From the viewpoint of enhancing the imaging ability of the compound or salt thereof according to the present invention in X-ray CT or fluorescent X-ray CT and/or from the viewpoint of increasing the lipid solubility of the compound or salt thereof according to the present invention and improving the permeability of the cell membrane The group represented by the above formula (YII) is preferably a group containing two or more iodine atoms, and particularly preferably a group represented by the following formula (YIII). In formula (YIII) below, R ³ represents an optionally substituted monovalent alkyl group or an optionally substituted monovalent halogenated alkyl group. Moreover, * represents a bond with an adjacent atom.

In one preferred aspect, the halogenated alkyl group in formula (YIII) is a 2-fluoroethyl group. In other words, in a preferred embodiment, Y in formula (I) is a group represented by formula (YIV) below. As shown in Experimental Examples below, the intracellular boron concentration can be dramatically increased by using a boron-containing compound containing a group represented by the following formula (YIV).

In addition, the compound of the present invention has a linker L between X and Y. As used herein, the term "linker" refers to a group that connects X, which is a boron-containing group, and Y, which is an iodine-containing group. can be of any kind. For example, it may be a divalent group having a basic skeleton composed of one or more atoms selected from carbon, oxygen, sulfur and nitrogen atoms. More preferably, it may be a divalent group having a heterocyclic ring composed of two or more atoms selected from carbon, oxygen, sulfur and nitrogen atoms.

More specifically, L is, for example, -O-, -S-, -NH-, -CO-, -CH ₂ -, -CH ₂ O-, -OCH ₂ -, -(CH ₂ O) _n -, -NHCO-, -CONH-, -OCO-, -COO-, -SCO-, -COS-, -NHCONH-, -NHCOO-, -SCH ₂ CONH-, -SCH ₂ COO-, -Ph-, -OPh-, -PhO-, -SPh-, -PhS-, -NHPh-, -PhNH-, -heterocycle-, -O-heterocycle-, -heterocycle-O-, -O-heterocycle-O -, -S-heterocycle-, -heterocycle-S-, -S-heterocycle-S-, -NH-heterocycle-, -heterocycle-NH-, -NH-heterocycle-NH-, -S -heterocycle-O-, -S-heterocycle-NH-, -O-heterocycle-S-, -O-heterocycle-NH-, -NH-heterocycle-S-, -NH-heterocycle-O - and the like, but are not limited to these. In the above formula, Ph represents 1,2-phenylene, 1,3-phenylene or 1,4-phenylene.

As described above, in one preferred embodiment, X in formula (I) is a monovalent group consisting of a boron cluster, and typical substances providing such groups include dodecaborate ([B ₁₂ H ₁₂ ] ^2- ), which has a thiol group, BSH ([B ₁₂ H ₁₁ SH] ^2- ), compound 2) is known. Therefore, in one preferred aspect, L in formula (I) is a group derived from a thiol group and a thiol-reactive functional group. Such groups include thioether-containing groups, thioester-containing groups, or disulfide-containing groups. More specific examples include, but are not limited to, groups containing thioethers as described in formula (LI) below.

Specific examples of the compound represented by formula (I) having X, Y, and L described above include, but are not limited to, compounds 3a to 3c shown in experimental examples described later.

The type of salt of the compound of the present invention is not particularly limited, and examples thereof include alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as calcium salts and magnesium salts, and metal salts such as aluminum salts. ammonium salts such as ammonium salts, tetramethylammonium salts and tetrabutylammonium salts, and salts with organic bases such as trimethylamine, triethylamine and ethylenediamine. However, it is not limited to these, and basically any pharmaceutically acceptable salt may be used.

A composition comprising a compound of the invention or a salt thereof may be provided as a pharmaceutical composition, particularly for boron neutron capture therapy. Accordingly, the present invention also encompasses in another aspect the use of a compound according to the present invention or a salt thereof for manufacturing a pharmaceutical composition, in particular a pharmaceutical composition for boron neutron capture therapy.

The compounds or salts thereof according to the present invention and pharmaceutical compositions containing them are particularly suitable for treating tumor diseases. The type of tumor disease to which the boron neutron capture therapy using the pharmaceutical composition according to the present invention is applied is not particularly limited. glioblastoma, etc.), malignant melanoma, head and neck cancer, lung cancer, liver cancer, thyroid cancer, kidney cancer, prostate cancer, breast cancer, pancreatic cancer, colon cancer, bladder cancer, meningioma, mesothelioma, sarcoma , gastric cancer, ovarian cancer, uterine cancer, cervical cancer, pharyngeal cancer, laryngeal cancer, etc.

There is no particular limitation on the administration method of the compound or salt thereof according to the present invention, and the pharmaceutical composition containing them, and an appropriate administration method may be selected according to the subject of application. Examples include, but are not limited to, oral administration, sublingual administration, intravenous administration, intraarterial administration, intraperitoneal administration, intramuscular administration, subcutaneous administration, topical administration including intratumoral administration, drip administration, and the like.

The dosage form of the pharmaceutical composition according to the present invention is not particularly limited, and may be an appropriate dosage form depending on the application target and administration method. For example, dosage forms such as injections, tablets, powders, granules, syrups, and capsules can be used, but are not limited to these. Moreover, it is good also as a formulation of a just before use type. The pharmaceutical composition according to the present invention may further contain pharmaceutically acceptable excipients, solvents, binders, stabilizers, dispersants, etc., depending on its dosage form and administration method. Needless to say.

Moreover, the pharmaceutical composition according to the present invention may further contain a drug delivery agent. Here, the drug delivery agent means pharmacokinetics of a compound or a salt thereof, which is an active ingredient of a pharmaceutical composition containing the agent, more specifically, retention in blood, distribution to target tissues, or Refers to a chemical substance that can increase permeability compared to the case without drug delivery agent. There are no particular restrictions on the type of drug delivery agent to be compounded in the pharmaceutical composition according to the present invention, but examples include lipids, vitamins, carbohydrates, proteins, antibodies, peptides, peptidomimetics, nucleic acids, nucleic acid mimetics, and polyamines. , synthetic or natural polymers or oligomers, microparticles, and combinations thereof. Regarding drug delivery agents used for boron-containing compounds, for example, "Hiroshi Fukuda," Neutron capture therapy (BNCT) for cancer using boron compounds, Tohoku Pharmaceutical University Research Journal, 62, 1-11 (2015), "Hiroyuki Nakamura Written "Boron Neutron Capture Therapy: Next Generation Radiation Therapy to Generate Alpha Rays in Cancer Cells, Drug Delivery System 35-2, 2020" "Michiue H, et al. Journal of Controlled Release, 330, 788-796 (2021 )”, etc.

In one preferred aspect, the drug delivery agent contained in the pharmaceutical composition of the present invention is a cell permeable peptide. Cell penetrating peptides are peptides that have the property of penetrating the cell membrane of cells, particularly human cells.

In one preferred aspect, the cell-permeable peptide contained in the pharmaceutical composition of the present invention can be a peptide containing hydrophobic amino acid residues and basic amino acid residues. As a peptide containing a hydrophobic amino acid residue and a basic amino acid residue, for example, AAAAAAK described in Patent Document 1 and Non-Patent Document 2 (in this specification, it may be referred to as "A6K". Note that A represents L-alanine, K represents L-lysine.) and AAAAAAR (in this specification, sometimes referred to as "A6R". A represents L-alanine and R represents L-arginine.) can be done. As shown in the experimental examples described later, the combination of the compound of the present invention or a salt thereof and a cell-permeable peptide containing a hydrophobic amino acid residue and a basic amino acid residue dramatically increases the intracellular boron concentration. be able to. When used in combination, the ratio of the compound according to the present invention or a salt thereof to the cell-penetrating peptide containing a hydrophobic amino acid residue and a basic amino acid residue can be set as appropriate, but usually the molar ratio is 20: A range of 1 to 1:10, preferably 10:1 to 1:1, more preferably 5:1 to 1:1 can be exemplified. Here, the numerical range indicated by "-" means the numerical range including the upper limit and the lower limit.

The compounds according to the present invention or salts thereof, and pharmaceutical compositions containing them are suitably used for boron neutron capture therapy. Accordingly, in another aspect, the present invention also includes a method of treating tumor disease, comprising the step of administering to a patient a pharmaceutical composition comprising a compound of the present invention or a salt thereof. The patient to whom the tumor disease treatment method according to the present invention is applied may be a human or a non-human animal suffering from a tumor disease.

The method for treating a tumor disease according to the present invention comprises, for example, administering a pharmaceutical composition containing a compound or a salt thereof according to the present invention to a patient suffering from a tumor disease, followed by treatment with low-energy radiation such as thermal neutron beams or epithermal neutron beams. It is performed by irradiating the patient with neutron beams. Thus, in one preferred aspect, the method for treating tumor disease according to the present invention comprises the steps of administering a compound of the present invention or a salt thereof to a patient, and irradiating the patient with neutron beams. , can be a method of treating tumor diseases.

Irradiation with low-energy neutron beams such as thermal neutron beams or epithermal neutron beams is preferably performed after the compound of the present invention or a salt thereof reaches the site to be treated, for example, administration of the compound of the present invention or a salt thereof. and after a predetermined period of time has passed. However, the compound or salt thereof of the present invention does not necessarily need to be administered prior to irradiation with low-energy neutron beams, and may be administered during irradiation with low-energy neutron beams. may be irradiated with low-energy neutron beams while administering As will be described later, the degree of accumulation of the compound of the present invention or its salt in the site to be treated can be determined by measuring the iodine atom concentration by X-ray CT, fluorescent X-ray CT, or the like.

There is no particular limitation on the irradiation method of the low-energy neutron beam, and irradiation may be performed by an appropriate method according to the type of tumor to be treated, the position of the site to be treated, and the like. For example, if the tumor to be treated is exposed on the surface, such as skin cancer, the site may be directly irradiated with low-energy neutron beams. On the other hand, if the tumor to be treated is not exposed on the surface, the low-energy neutron beam may be irradiated through the skin, or the area may be completely or partially exposed and the low-energy neutron beam is irradiated. You may There are no particular restrictions on the type of low-energy neutron beam to be irradiated, but for example, when treating a tumor located deep in the body, epithermal neutron beams, which change into thermal neutrons in vivo, are used as low-energy neutron beams. Epithermal neutron beams with an energy range of about 0.6 to 40 keV are preferably used.

In addition, the number of times of neutron beam irradiation may be one or more times, depending on the site and condition of the tumor disease to be treated, or taking into account the subject's age, sex, treatment period, etc. Then, the medical staff can select the appropriate number of times.

The neutron beam generation source is not particularly limited, and a nuclear reactor may be used, an accelerator such as a cyclotron, a linear accelerator, an electrostatic accelerator may be used, or a combination thereof may be used.

Moreover, the method for treating tumor diseases according to the present invention may be performed as a single treatment, or may be used in combination with surgery and/or chemotherapy.

On the other hand, the compound containing boron and iodine or a salt thereof contains iodine that functions as a contrast agent in X-ray CT in addition to boron, so the amount of boron accumulated in the tumor or the distribution state is measured more than boron. It can be understood by measuring the accumulated amount or distribution of iodine in the tumor and converting the obtained iodine concentration to the boron concentration. Thus, in another aspect of the present invention, the step of administering a compound containing boron and iodine or a salt thereof to a patient, the step of measuring the iodine concentration in the patient's tumor, the measured iodine concentration A method for quantifying boron concentration in a tumor comprising estimating the boron concentration in a tumor of said patient based on: The compound containing boron and iodine may be the compound according to the present invention or a salt thereof as shown in [1] to [8] above. Also, the patient can be a human or non-human animal. The type of tumor, the method of administering the compound, etc. are the same as those already described for the method of treating tumor diseases according to the present invention.

Needless to say, the method for treating tumor disease according to the present invention may include the method for quantifying the boron concentration. That is, in a preferred aspect of the method for treating a tumor disease according to the present invention, the steps of administering a compound of the present invention or a salt thereof to a patient, measuring the iodine concentration in the patient's tumor, A method for treating a tumor disease, comprising the steps of estimating the boron concentration in the patient's tumor based on the iodine concentration, and irradiating the patient with neutron beams based on the estimated boron concentration. Irradiating the patient with a neutron beam based on the estimated boron concentration means that the neutron beam irradiation conditions such as the type, intensity, irradiation time, number of times, and irradiation direction of the neutron beam to be irradiated to the patient are set to the estimated boron concentration. and irradiate the patient with neutron beams according to the set irradiation conditions.

Although any method may be used to measure the iodine concentration, for example, X-ray CT can be used. Moreover, not only X-ray CT but also fluorescent X-ray CT may be used. According to fluorescent X-ray CT, the distribution of iodine in vivo, including tumor tissue, can be imaged and/or quantitatively evaluated with superior spatial resolution.

The intracellular iodine concentration measured by an appropriate method can be converted into an intracellular boron concentration, for example, according to the formula shown below. In the formula below, "the number of iodine atoms per molecule" means the number of iodine atoms contained in one molecule of a compound containing boron and iodine or a salt thereof to be administered to a patient. "Number of boron atoms per" means the number of iodine atoms contained in one molecule of a boron and iodine containing compound or salt thereof administered to a patient.

Thus, by using the compound or salt thereof according to the present invention, the boron concentration in the living body, which was conventionally difficult to measure, can be accurately grasped based on the measured value of the iodine concentration, which is easier to measure than the boron concentration. becomes possible. Therefore, irradiation conditions such as the dose of boron-containing compounds to be administered to patients and the dose of neutron beams to be irradiated to patients can be set more appropriately, thereby improving the therapeutic effect of boron neutron capture therapy and reducing side effects. can be expected.

The iodine atom contained in the compound or salt thereof according to the present invention may be iodine-123 ( ¹²³ I). When the compound or salt thereof according to the present invention contains ¹²³ I, iodine or the present The amount of the compound according to the invention or a salt thereof accumulated in tumor tissue may be measured and visualized using SPECT (Single Photon Emission Computed Tomography).

Moreover, when the compound or its salt which concerns on this invention is a compound containing fluorine, or its salt, the fluorine atom may be ^18F . When the compound of the present invention or its salt contains ¹⁸ F, PET (Positron Emission Tomography) may be used to measure the amount of accumulation of the compound of the present invention or its salt in tumor tissue.

The disclosures of all documents cited herein are hereby incorporated by reference in their entirety. Also, throughout this specification, where the singular forms of the words “a,” “an,” and “the” are included, the singular as well as the plural unless the context clearly indicates otherwise. shall include things.

Hereinafter, the present invention will be described in more detail with reference to specific examples, but the scope of the present invention is not limited to these specific examples.

(Experiment 1) Synthesis of compound

(Experiment 1-0) Description of method and equipment In the synthesis reactions shown below, the progress of all reactions was monitored on a 0.2 mm thick TLC plate (manufactured by Merck; glass back; product name: TLC glass plate silica gel 60 F ₂₅₄ '') was used to monitor.
Silica gel 60 (particle size 0.035-0.070 mm) was used for flash column chromatography.
¹ H NMR and ¹³ C NMR spectra were measured at room temperature using a Varian VXR-400 spectrometer ( ¹ H 400 MHz, ¹³ C 100 MHz). NMR solvents used were deuterated chloroform (CDCl ₃ ), methanol-d ₄ (CD ₃ OD), or acetonitrile-d ₃ (CD ₃ CN), as described below. In addition, the chemical shifts are the peaks of the deuterated solvent (CDCl ₃ : ¹ H δ 7.26 ppm, ¹³ C δ 77.11 ppm, CD ₃ OD: ¹ H δ 3.31 ppm, ¹³ C δ 49.00 ppm, CD ₃ CN: ¹ H δ 1.94 ppm, ¹³ C δ 1.32 and 118.26 ppm). The unit of coupling constant is Hz.
Melting points were measured with a hot stage melting point measuring apparatus manufactured by Yanagimoto Seisakusho, and are indicated without correction.

In addition, the purity of the compound synthesized in each step was confirmed by analytical HPLC. The HPLC system used in this experiment is a Shimadzu Corporation liquid chromatography system consisting of an LC-20AT pump, SPD-20A UV-visible spectrophotometer, CTO-10ASvp column oven, and LabSolutions software. Samples (20 μL each) were loaded onto an Inertsil ODS-3 column (4.6 i.d.×100 mm, 3 μm, GL Sciences) equipped with a guard column at 40° C. with phosphate buffer (pH 2) as the mobile phase. injected. The flow rate was 0.7 mL/min and the absorbance at 230 nm was monitored. All solvents were of analytical grade and used without further purification. Purity of all compounds tested was greater than 95% as confirmed by HPLC.

(Experiment 1-1) Synthesis of 2,6-diiodo-4-nitrophenol (compound 5)

The synthesis of 2,6-diiodo-4-nitrophenol (compound 5) was described by Thomas M.; Beale et al. ACS. Med. Chem. Lett. 2012, 3, 177-181. was performed according to the method described in . Briefly, sodium chlorite (approximately 80%, 1.6 g, 14 mmol) and sodium iodide (4.3 g, 29 mmol) were dissolved in H ₂ O (100 mL) and the solution was cooled on an ice bath. It was cooled (the solution is called "solution A"). Next, 4-nitrophenol (1.0 g, 7.19 mmol, compound 4) was dissolved in methanol (100 mL) and cooled on an ice bath (this solution is called "solution B"). After adding solution B and concentrated hydrochloric acid (1.34 mL) to solution A on an ice bath under an argon atmosphere, the reaction mixture was stirred at room temperature for 24 hours. The reaction mixture was adjusted to pH 3 and extracted with ethyl acetate (100 mL x 2). The organic layer was collected and washed with saturated aqueous Na ₂ S ₂ O ₃ (50 mL). Further, the organic layer was washed with H ₂ O (50 mL×2), brine (50 mL) and evaporated under reduced pressure. Then, it was recrystallized from ethyl acetate to obtain compound 5 (2.57 g, 7.06 mmol, yield 98%) as a yellow solid. Further, yellow needle crystals of Compound 5 were obtained by recrystallization from ethyl acetate. ¹ H NMR measurement results of the obtained compound 5 are as follows: ¹ H NMR (CDCl ₃ , 400 MHz) δ: 8.60 (2H, s), 6.38 (1H, s).

(Experiment 1-2) Synthesis of 1,3-diiodo-2-methoxy-5-nitrobenzene (compound 6a)

Synthesis of 1,3-diiodo-2-methoxy-5-nitrobenzene (compound 6a) was reported by Thomas M.; Beale et al. ACS. Med. Chem. Lett. 2012, 3, 177-181. was performed according to the method described in . That is, 2,6-diiodo-4-nitrophenol (compound 5) (1.35 g, 3.46 mmol) was dissolved in acetone (20 mL), and potassium carbonate (1.43 g, 10.4 mmol) was added. and dimethyl sulfate (0.98 mL, 10.4 mmol) were added. The reaction mixture was refluxed for 19 hours. Aqueous ammonia (28% in water, 10 mL) was added to the reaction mixture on an ice bath and stirred for 10 minutes. The reaction mixture was extracted with ethyl acetate (100 mL x 2). The organic layer was collected, washed with brine (50 mL), dried over MgSO ₄ and evaporated under reduced pressure to give compound 6a (1.26 g, 3.12 mmol, 91% yield) as a white Obtained as a solid. ¹ H NMR measurement results of the obtained compound 6a are as follows: ¹ H NMR (CDCl ₃ , 400 MHz) δ: 8.64 (2H, s), 3.94 (3H, s).

(Experiment 1-3) Synthesis of 1,3-diiodo-2-ethoxy-5-nitrobenzene (compound 6b)

Synthesis of 1,3-diiodo-2-ethoxy-5-nitrobenzene was carried out according to the method for synthesizing compound 6a described above. That is, compound 5 (586 mg, 1.50 mmol) was dissolved in acetone (10 mL), and potassium carbonate (622 mg, 4.50 mmol) and diethyl sulfate (588 μL, 4.50 mmol) were added. The reaction mixture was refluxed for 18 hours. Aqueous ammonia (28% in water, 10 mL) was added to the reaction mixture on an ice bath and stirred for 10 minutes. The reaction mixture was extracted with EtOAc (50 mL x 2). The organic layer was collected, washed with brine ₍ 50 mL), dried over MgSO4 and evaporated under reduced pressure to give 658 mg of solid. This solid was purified by chromatography using an eluent of EtOAc/n-Hexane=1/20 to give compound 6b (534 mg, 1.27 mmol, 85% yield) as a yellow solid. ¹ H NMR measurement results of the obtained compound 6b are as follows: ¹ H NMR (CDCl ₃ , 400 MHz) δ: 8.64 (2H, s), 4.13 (2H, q, J=6 .8 Hz), 1.58 (3H, t, J=6.8 Hz).

(Experiment 1-4) Synthesis of 4-amino-2,6-diiodophenol (compound 7)

4-Amino-2,6-diiodophenol (compound 7) was synthesized by a reduction reaction of the nitro group of 2,6-diiodo-4-nitrophenol (compound 5). That is, compound 5 (1.17 g, 3.00 mmol) was dissolved in a mixed solution of ethanol (15 mL) and ethyl acetate (15 mL), then tin(II) chloride (2.84 g, 15.0 mmol) was added. The reaction mixture was stirred at 70° C. for 18 hours. NaHCO ₃ saturated aqueous solution (20 mL) was added to the reaction mixture and filtered through celite. The filtrate was extracted with ethyl acetate (50 mL x 2). The organic layer was collected, washed with brine ( ₄₀ mL), dried over MgSO4, evaporated under reduced pressure and recrystallized from ethyl acetate to give compound 7 (883 g, 2.45 mmol). , yield 82%) as a yellow solid. ¹ H NMR measurement results of the obtained compound 7 are as follows: ¹ H NMR (CDCl ₃ , 400 MHz) δ: 7.15 (2H, s).

(Experiment 1-5) Synthesis of 1,3-diiodo-2-methoxy-5-aminobenzene (compound 8a)

Synthesis of 1,3-diiodo-2-methoxy-5-aminobenzene (compound 8a) was carried out by reducing the nitro group of 1,3-diiodo-2-methoxy-5-nitrobenzene (compound 6a). Compound 6a (802 mg, 1.99 mmol) was dissolved in a mixed solution of ethanol (10 mL) and ethyl acetate (10 mL), then tin(II) chloride (1.886 g, 9.95 mmol) was added. rice field. The reaction mixture was stirred at 70° C. for 6.5 hours. NaHCO ₃ saturated aqueous solution (20 mL) was added to the reaction mixture and filtered through celite. The filtrate was extracted with ethyl acetate (40 mL x 2). The organic layer was collected, washed with brine (50 mL×2), dried over MgSO ₄ , evaporated under reduced pressure and recrystallized from ethyl acetate to give compound 8a (756 mg, 1. 99 mmol, q.y.) as a solid. The obtained compound 8a was directly used in the next step.

(Experiment 1-6) Synthesis of 1,3-diiodo-2-ethoxy-5-aminobenzene (compound 8b)

Synthesis of 1,3-diiodo-2-ethoxy-5-aminobenzene (compound 8b) was carried out by reducing the nitro group of 1,3-diiodo-2-ethoxy-5-nitrobenzene (compound 6b). That is, compound 6b (419 mg, 1.0 mmol) was dissolved in a mixed solution of ethanol (5 mL) and ethyl acetate (5 mL), then tin(II) chloride (948 mg, 5.0 mmol) was added. rice field. The reaction mixture was stirred at 70° C. for 2.5 hours. NaHCO ₃ saturated aqueous solution (10 mL) was added to the reaction mixture and filtered through celite. The filtrate was extracted with ethyl acetate (40 mL x 2). The organic layer was collected, washed with brine (50 mL×2), dried over MgSO ₄ , evaporated under reduced pressure and recrystallized from ethyl acetate to give compound 8b (376 mg, 0.5 m). 97 mmol, 97% yield) as a solid. The obtained compound 8b was directly used in the next step.

(Experiment 1-7) Synthesis of 2-(2-fluoroethoxy)-1,3-diiodo-5-aminobenzene (compound 8c)

Compound 7 (310 mg, 0.86 mmol) was dissolved in acetone (9 mL), potassium carbonate (238 mg, 1.72 mmol) and 1-fluoro-2-iodoethane (83 μL, 1.03 mmol) were added. added. The reaction mixture was stirred at room temperature for 21 hours. Water (50 mL) was poured into the reaction mixture and extracted with ethyl acetate (40 mL x 3). The organic layer was collected, washed with water (30 mL×2), dried over MgSO ₄ and evaporated under reduced pressure to give compound 8c (306 mg, 0.75 mmol, 87% yield) as a green obtained as a solid. The measurement results of ¹ H NMR and ¹³ C NMR of the obtained compound 8c are as follows: ¹ H NMR (CD ₃ OD, 400 MHz) δ: 7.14 (2H, s), 4.77 (2H , dt, J=47.6, 4.0 Hz), 4.12 (2H, dt, J=47.6, 4.0 Hz); ¹³ C NMR (CD ₃ OD, 100 MHz) δ: 148. 36, 147.28, 125.13, 89.48, 83.06, 81.37, 71.97, 71.77.

(Experiment 1-8) Synthesis of 1-(3,5-diiodo-4-methoxyphenyl)-1H-pyrrole-2,5-dione (compound 9a)

The synthesis of 1-(3,5-diiodo-4-methoxyphenyl)-1H-pyrrole-2,5-dione (compound 9a) was reported by Hongjuan Tong et al. Chem. Common. , 2017, 53, 3583-3586. was performed according to the procedure described in First, maleic anhydride (235 mg, 2.40 mmol) was added to an acetone solution (25 mL) of compound 8a (781 mg, 2.00 mmol). The reaction mixture was stirred at 30° C. for 6.5 hours and then evaporated under reduced pressure. Then sodium acetate (197 mg, 2.40 mmol) and acetic anhydride (25 mL) were added, and the mixture was stirred at 75° C. for 15 hours under an argon atmosphere. The reaction mixture was cooled to room temperature and the precipitated solid was collected. This solid was purified by chromatography using an eluent of EtOAc/n-Hexane=1/5 and recrystallized from ethyl acetate to give compound 9a (634 mg, 1.39 mmol, yield 70%). Obtained as a yellow solid. The measurement results of ¹ H NMR and ¹³ C NMR of the obtained compound 9a are as follows: ¹ H NMR (CDCl ₃ , 400 MHz) δ: 7.78 (2H, s), 6.86 (2H, ^13C NMR ( _CDCl3 , 100 MHz) ?: 168.88, 158.74, 137.12, 134.47, 129.18, 89.94, 60.s), 3.88 (3H, s); 92.

(Experiment 1-9) Synthesis of 1-(3,5-diiodo-4-ethoxyphenyl)-1H-pyrrole-2,5-dione (compound 9b)

1-(3,5-Diiodo-4-ethoxyphenyl)-1H-pyrrole-2,5-dione was synthesized according to the method for synthesizing compound 9a described above. Briefly, maleic anhydride (114 mg, 1.17 mmol) was added to an acetone solution (10 mL) of compound 8b (376 mg, 0.97 mmol). The reaction mixture was stirred at room temperature for 14 hours and then evaporated under reduced pressure. Then sodium acetate (96 mg, 1.17 mmol) and acetic anhydride (10 mL) were added. The mixture was stirred at 75° C. for 24 hours under an argon atmosphere. The reaction mixture was cooled to room temperature, and the precipitated solid was collected to obtain compound 9b (318 mg, 0.68 mmol, yield: 70%) as a white solid. The measurement results of ¹ H NMR and ¹³ C NMR of the obtained compound 9b are as follows: ¹ H NMR (CDCl ₃ , 400 MHz) δ: 7.77 (2H, s), 6.86 (2H, s), 4.06 (2H, q, J=7.0 Hz), 1.54 (3H, t, J=7.0 Hz); ¹³ C NMR (CDCl ₃ , 100 MHz) δ: 168.75 , 157.86, 136.90, 134.29, 128.76, 90.24, 69.48, 15.47.

(Experiment 1-10) Synthesis of 1-(4-(2-fluoroethoxy)-3,5-diiodophenyl)-1H-pyrrole-2,5-dione (compound 9c)

Maleic anhydride (0.11 g, 1.17 mmol) was added to an acetone solution (8 mL) of compound 8c (293 mg, 0.72 mmol). The reaction mixture was stirred at room temperature for 16 hours and then evaporated under reduced pressure to give 389 mg of a green solid. Then sodium acetate (70 mg, 0.86 mmol) and acetic anhydride (8 mL) were added. The mixture was stirred at 75° C. for 2 hours under an argon atmosphere. The reaction mixture was cooled to room temperature and the precipitated solid was collected to give compound 9c (241 mg, 0.495 mmol, yield 69%) as a white solid. ¹ H NMR measurement results of the obtained compound 9c are as follows: ¹ H NMR (CDCl ₃ , 400 MHz) δ: 7.81 (2H, s), 6.87 (2H, s), 4 .88 (2H, dt, J=4.0, 4.7 Hz), 4.28 (2H, dt, J=4.0, 4.7 Hz).

(Experiment 1-11) Synthesis of Compound 3a (BS-DIP-OMe)

Compound 9a (455 mg, 1.00 mmol) was dissolved in chloroform (10 mL) and acetonitrile (10 mL), magnesium sulfate (10 mg) was added, followed by n-BSH (220 mg, 1.00 mmol). rice field. The reaction mixture was stirred at 60° C. for 72 hours. After filtration, the solvent was removed under reduced pressure and the residue was purified by chromatography using an eluent of EtOAc/n-Hexane=1/5 to give compound 3a (439 mg, 0.65 mmol, 65% yield). was obtained as a yellow solid. The measurement results of ¹ H NMR of the obtained compound 3a are as follows: ¹ H NMR (CD ₃ CN, 400 MHz) δ: 7.78 (2H, s), 3.90 (1H, dd, J = 8.0, 4.0 Hz), 3.85 (3H, s), 3.25 (1H, dd, J = 19.0, 8.6 Hz), 3.11 (1H, dd, J = 19.0, 4.0 Hz), 1.80-0.50 (11H, br). The purity of compound 3a measured by HPLC was 97% (HPLC measurement conditions: eluent 20 mM phosphate buffer/H ₂ O = 50/50; column: ODS-3; detection wavelength: 230 nm; temperature: 40°C; hold time 2.566 min).

(Experiment 1-12) Synthesis of compound 3b (BS-DIP-OEt)

Compound 9b (305 mg, 0.65 mmol) was dissolved in chloroform (5 mL) and acetonitrile (5 mL), magnesium sulfate (10 mg) was added, followed by n-BSH (143 mg, 0.65 mmol). rice field. The reaction mixture was stirred at 60° C. for 38 hours. After filtration, the solvent was removed under reduced pressure and the residue was purified by chromatography using an eluent of EtOAc/n-Hexane=1/5 to give compound 3b (240 mg, 0.35 mmol, 54% yield). was obtained as a white solid. Also, it was recrystallized from EtOH/DCM to obtain white needle crystals. ¹ H NMR measurement results of the obtained compound 3b are as follows: ¹ H NMR (CD ₃ CN, 400 MHz) δ: 7.75 (2H, s), 4.03 (2H, q, J = 7.0 Hz), 3.88 (1H, dd, J = 8.8, 4.0 Hz), 3.25 (1H, dd, J = 16.0, 8.8 Hz), 3.07 (1H, dd, J=16.0, 4.0 Hz), 1.46 (3H, t, J=7.0 Hz), 1.70-0.50 (11H, br). In addition, the purity of compound 3b measured by HPLC was 96% (HPLC measurement conditions: eluent 10 mM phosphate buffer; column: ODS-3; detection wavelength: 230 nm; temperature: 40 ° C.; retention time: 8.893 minutes). The melting point of the obtained compound 3b was 252.1-263.5°C.

(Experiment 1-13) Synthesis of Compound 3c (BS-DIP-OEF)

Compound 9c (213 mg, 0.436 mmol) was dissolved in chloroform (4.36 mL) and acetonitrile (4.36 mL), sodium sulfate (310 mg, 2.18 mmol), n-BSH (92 mg, 0.436 mmol) was added. The reaction mixture was stirred at 60° C. for 17 hours. After filtration, the solvent was removed under reduced pressure and the residue was purified by chromatography using an eluent of MeOH/DCM=1/8 to give compound 3c (113 mg, 0.16 mmol, yield: 37%). Obtained as a white solid. The measurement results of ¹ H NMR of the obtained compound 3c are as follows: ¹ H NMR (CD ₃ CN, 400 MHz) δ: 7.80 (2H, s), 4.85 (2H, dt, J = 47.0, 4.0 Hz), 4.27 (2H, dt, J = 30.0, 4.0 Hz), 3.92 (1H, dd, J = 8.6, 4.0 Hz) , 3.27 (1H, dd, J = 19.0, 8.6 Hz), 3.14 (1H, dd, J = 19.0, 4.0 Hz), 1.70-0.50 (11H , br). In addition, the purity of compound 3b measured by HPLC was 95% (HPLC measurement conditions: eluent acetonitrile/sodium perchlorate (50 mM) buffer = 65/35; column: ODS-3; detection wavelength: 230 nm temperature: 40°C; hold time 1.446 min).

(Experiment 2) Measurement of intracellular uptake concentration

(Experiment 2-1) Preparation of Stock Solution A Containing Boron-Containing Compound 14.04 mg of Compound 3c (BS-DIP-OEF (F.W. 698.16)) was weighed into a 2 mL Eppendorf tube. Since compound 3c and other boron-containing compounds used in this experiment are highly hygroscopic, these compounds were weighed in a glove box adjusted to a nitrogen atmosphere and a humidity of 40% or less. In addition, before weighing compound 3c in the glove box, weigh (tare) an empty 2 mL Eppendorf tube in advance with a precision balance outside the glove box, and add compound 3c to the Eppendorf tube in the glove box. Weighed out. The weight of the Eppendorf tube containing the weighed compound 3c was measured with a precision balance outside the glove box, and the increase in weight before and after weighing was taken as the weight of the weighed compound 3c.

Then, 1005.5 μL of sterile Milli-Q water was added to the 2 mL Eppendorf tube in which compound 3c was measured to prepare a solution with a concentration of 20 mM of compound 3c. The resulting solution was placed in an ultrasonic water bath for 10 minutes and sterilized with a 0.2 μm sterilizing filter. A stock solution A was prepared by diluting the solution 2-fold with Milli-Q water so that the concentration of compound 3c was 10 mM.

Stock solutions A with a concentration of 10 mM were similarly prepared for compounds 3a, 3b and n-BSH following the procedure described above for compound 3c.

(Experiment 2-2) Preparation of Cell Treatment Solution Conventional boron cluster BSH (n-BSH) and boron-containing compounds (compounds 3a, 3b, 3c) were stocked at concentrations of 1,000 μM (1 mM). Solution A, Milli-Q water, and RPMI medium (RPMI-1640 (Sigma-Aldrich, catalog number: R8758), supplemented with 10% fetal bovine serum (FBS), 0.5% antibiotics (penicillin 50,000 U/L, streptomycin 50,000 μg/L)) was mixed to prepare cell treatment solutions 1 to 4. A cell treatment solution was prepared by adding stock solution A containing a boron-containing compound, Milli-Q water, and RPMI medium in this order to a 15 mL centrifuge tube. At this time, after mixing stock solution A and Milli-Q water, the mixture was placed in an ultrasonic water bath for 10 minutes while cooling with ice so as not to exceed 30° C., and then RPMI medium was added. 4 mL of each cell treatment solution was prepared. The amount of each solution added to prepare a total of 4 mL of cell treatment solution is as shown in Table 1 below.

(Experiment 2-3) Cell culture, compound treatment, preparation of cell lysate B16/BL6 cells, which are mouse melanoma cells (purchased from RIKEN, cell number: RCB2638), were placed in a 35 mm dish (Falcon cell culture dish, product number : 353001) so that the number of cells was 60×10 ⁴ cells/2 mL, and cultured at 37° C. in 5% CO ₂ for 48 hours. After that, 1 mL of the medium was removed from each dish, and 1 mL of the cell treatment solutions 1 to 4 prepared above was added. As shown in Table 1, the concentration of the boron-containing compound in the dishes to which Cell Treatment Solutions 2 to 4 were added was approximately 500 μM (0.5 mM). After incubating for 2 hours under conditions of 5% CO ₂ and 37° C., the medium in the dish was removed as much as possible with an aspirator, and cooled physiological saline (1 mL, manufactured by Otsuka Pharmaceutical Co., Ltd., the same applies hereinafter) was used. and washed in the usual manner. Thereafter, 1 mL of cooled physiological saline was added to each dish, and the cells were detached and suspended from each dish using a scraper. From the suspension thus obtained, 10 μL was taken, diluted 2-fold with trypan blue, and the number of cells was counted using a hemocytometer. On the other hand, for each dish, the remaining suspension was transferred to a 2 mL Eppendorf tube, centrifuged at 4° C. and 1,800 rpm for 3 minutes, and after centrifugation, the supernatant was removed. After that, RIPA Buffer (Fujifilm Wako Pure Chemical Industries, Ltd., catalog number: 182-02451) was added in 0.5 mL portions and pipetted several times. Incubate on ice for 10 minutes to lyse the cells and obtain a cell lysate. The resulting cell lysate was stored at 4°C. In addition, protein quantification of the obtained cell lysate was performed using BCA Protein Assay Kit (manufactured by Takara Bio Inc.).

Intracellular boron concentration and intracellular iodine concentration were measured by liquid chromatography-mass spectrometry (LC-MS/MS). Specific procedures are as follows.

First, the entire amount of the cell lysate obtained by the above-described procedure is taken in a 0.5 mL syringe, and filtered by attaching a syringe filter (Millex Syringe Filter (0.22 μm), manufactured by Merck Co., Ltd.). Obtained in .5 mL Eppendorf tubes. 20 μL of the obtained filtered sample was placed in another Eppendorf tube, and 180 μL of developing solvent for LCMS was added thereto to dilute 10-fold. 100 μL of the diluted sample was dispensed into an autosampler sample cup. The composition of the developing solvent is as follows: 5 mM DHAA acetonitrile solution/5 mM DHAA Milli-Q aqueous solution = 65/35; Acetonitrile purchased from Merck Co., Ltd. (product code: 114291) was used.

On the other hand, as a compound solution for the calibration curve, compounds 3a to 3c were dissolved in a developing solvent at 10,000, 1,000, 100, 10, 1, and 0.1 nM as a guide, and a dilution series for the calibration curve was prepared for each compound. made. If the compound concentration measured for each sample did not fall within the above range, an additional dilution series for the calibration curve was prepared so that the concentration fell within the concentration range of the dilution series for the calibration curve.

The LC-MS system was an API4000 LC-MS/MS system (Applied Biosystems Inc.) consisting of an LC-20AD pump, an SPD-20AV UV-Vis spectrophotometric detector, and a CTO-20AC column oven (Shimadzu Corporation). , Toronto, Ontario, Canada). The measurement conditions are as follows: developing solvent 5 mM DHAA acetonitrile solution/5 mM DHAA Milli-Q aqueous solution = 65/35; flow rate 0.2 mL/min; UV 230 nm; autosampler temperature 15°C; Oven temperature 40° C.; column Tosoh ODS-100V, 5 cm.

For each sample, the intracellular compound concentration was obtained based on the peaks of compounds 3a-3c obtained and the standard curve. Based on the obtained intracellular compound concentration, intracellular boron concentration and intracellular iodine concentration in each sample were obtained.

FIG. 2 shows the measurement results of the intracellular boron concentration of each sample obtained as described above. In FIG. 2, the measurement results are shown as the mean±standard deviation of the values obtained from three independent tests for each sample.

As shown in FIG. 2, compound 3a (cell treatment solution 2), compound 3b (cell treatment solution 3) and compound 3c (cell treatment solution 4) are all taken up into cells, and in particular, compared with compound 3a and compound 3b, As a result, fluorine-bearing compound 3c gave a significantly higher intracellular boron concentration of about 40 ppm. Boron neutron capture therapy (BNCT) requires an intracellular boron concentration of about 20 to 40 ppm, but it has been reported that even if BSH is used alone, an intracellular boron concentration of 20 to 40 ppm cannot be obtained. (For example, Non-Patent Document 2: Journal of Controlled Release, 330, 788-796 (2021)). In contrast, it is surprising that the fluorine-containing compound 3c gave intracellular boron concentrations as high as about 40 ppm even when used at a treatment concentration of 500 μM, which is significantly lower for a BNCT boron agent. This is a remarkable effect. From the above results, compound 3c was shown to have a particularly excellent effect of increasing the intracellular boron concentration.

(Experiment 3) Combined experiment with cell-permeable peptide As described above, by using compound 3c, a higher intracellular boron concentration could be achieved compared to compounds 3a and 3b and conventional boron cluster BSH. . Therefore, with respect to compound 3c, not only compound 3c is used alone, but also the cell-permeable peptide A6K peptide, which is reported to have the effect of increasing the intracellular uptake of boron clusters (for example, patent publication No. 6548060, Journal of Controlled Release, 330, 788-796 (2021)).

(Experiment 3-1) Preparation of stock solution B containing cell-penetrating peptide A6K A6K peptide hydrochloride (Ac-A6-K-NH ₂ ·HCl, custom synthesis by Biologica, FW = 613.71 + 36.46 = 650.17, A for L-alanine, K for L-lysine) (8.912 mg, 0.005 mmol) was weighed into a 2 mL Eppendorf tube. Since the A6K peptide hydrochloride easily generates static electricity, the A6K peptide hydrochloride was weighed on a medicine wrapping paper. 1370.72 μL of sterilized Milli-Q water was added to the 2 mL Eppendorf tube into which A6K hydrochloride was weighed out in a clean bench to give a solution with a concentration of 10 mM. When dissolving the A6K hydrochloride, an ultrasonic water bath was used to completely dissolve it. The resulting solution was sterilized with a 0.2 μm sterile filter. This is to avoid decomposition of the A6K peptide by bacteria or the like. The solution thus obtained was designated as stock solution B. Stock solution B was stored at 4° C. and subjected to an ultrasonic water bath again before use.

(3-2) Preparation of Cell Treatment Solution Stock solution A, stock solution B, Milli-Q water, and RPMI medium (RPMI-1640 (Sigma-Aldrich, catalog number: R8758), supplemented with 10% fetal bovine serum (FBS) so that μM/1,000 μmM , 0.5% antibiotics (penicillin 50,000 U/L, streptomycin 50,000 μg/L)) were mixed to prepare cell treatment solutions 5-7. For the preparation of cell treatment solutions 5 to 7, stock solution A containing a boron-containing compound (compound 3c), stock solution B containing A6K peptide, Milli-Q water, and RPMI medium were added in this order to a 15 mL centrifuge tube. It was done by adding At this time, stock solution A, stock solution B, and Milli-Q water were mixed, cooled with ice so as not to exceed 30° C., placed in an ultrasonic water bath for 10 minutes, and then RPMI medium was added. 4 mL of each cell treatment solution was prepared. The amount of each solution added to prepare a total of 4 mL of cell treatment solution is as shown in Table 2 below. The methods for preparing cell-treated solution 1 and cell-treated solution 4, which are comparative objects, are extracted from Table 1 and shown again.

(Experiment 3-3) Cell culture/compound treatment/cell lysate preparation In the same procedure as shown in (Experiment 2-3), cell culture, cell treatment with cell treatment solution, and cell lysate preparation were performed. went. That is, B16/BL6 cells (purchased from RIKEN, cell number: RCB2638), which are mouse melanoma cells, were placed in a 35 mm dish (Falcon cell culture dish, product number: 353001) and the number of cells was 60 × 10 ⁴ cells/2 mL. and cultured for 48 hours under conditions of 5% CO ₂ and 37°C. After that, 1 mL of medium was removed from each dish, and 1 mL of cell treatment solution 1, 2 or 5 to 7 was added. As shown in Table 2, the concentration of the boron-containing compound in the dish to which Cell Treatment Solution 2 or 5-7 was added was approximately 500 μM (0.5 mM). After incubating for 2 hours under conditions of 5% CO ₂ and 37° C., the medium in the dish was removed as much as possible with an aspirator, and cooled physiological saline (1 mL, manufactured by Otsuka Pharmaceutical Co., Ltd., the same applies hereinafter) was used. and washed in the usual manner. Thereafter, 1 mL of cooled physiological saline was added to each dish, and the cells were detached and suspended from each dish using a scraper. From the suspension thus obtained, 10 μL was taken, diluted 2-fold with trypan blue, and the number of cells was counted using a hemocytometer. On the other hand, for each dish, the remaining suspension was transferred to a 2 mL Eppendorf tube, centrifuged at 4° C. and 1,800 rpm for 3 minutes, and after centrifugation, the supernatant was removed. After that, RIPA Buffer (Fujifilm Wako Pure Chemical Industries, Ltd., catalog number: 182-02451) was added in 0.5 mL portions and pipetted several times. Incubate on ice for 10 minutes to lyse the cells and obtain a cell lysate. The resulting cell lysate was stored at 4°C. In addition, protein quantification of the obtained cell lysate was performed using BCA Protein Assay Kit (manufactured by Takara Bio Inc.).

Intracellular boron concentration and intracellular iodine concentration were measured using liquid chromatography-mass spectrometry (LC-MS/MS) in the same procedure as shown in (Experiment 2-3). Table 3 shows the results obtained.

As shown in Table 3, when compound 3c was used alone (cell treatment solution 4), the intracellular boron concentration after incubation for 2 hours at a concentration of 500 μM of compound 3c was 39±13 ppm, 25 μM, 50 μM. , 100 μM A6K peptide increased the intracellular boron concentration to 57±4 ppm, 63±38 ppm, 402±103 ppm after incubation under the same conditions. This is well above the intracellular boron concentration of 40 ppm normally required for boron neutron capture therapy mentioned above. According to Non-Patent Document 2, when BSH (500 μM), which is a conventional boron cluster, and A6K peptide (100 μM) are used together, the intracellular boron concentration is about 2,000 ng/10 ⁶ cells. (Non-Patent Document 2, Figure 2B). In contrast, when compound 3c (500 μM) and A6K peptide (100 μM) were used in combination, the intracellular boron concentration was 3200±828 ng/10 ⁶ cells, which is approximately 1 It was found that an intracellular boron concentration as high as 0.6 times was obtained. It was shown that the intracellular boron concentration can be remarkably increased by using a compound containing boron and iodine in combination with a cell-permeable peptide.

According to the compound or its salt according to the present invention, and the pharmaceutical composition containing these, the boron concentration in the affected area can be obtained quantitatively by measuring the iodine concentration, which is easier to measure, instead of the boron concentration. Since it is possible, it is now possible to easily obtain information about the boron concentration in the affected area, which was very difficult to obtain, and in a preferred embodiment, the compound or salt thereof according to the present invention, and these can achieve higher intracellular boron concentrations compared to conventional boron-containing compounds. Therefore, the present invention is expected to improve the therapeutic effect of boron neutron capture therapy and reduce side effects. Thus, the present invention contributes to the application of boron neutron capture therapy in the treatment of neoplastic diseases, and has great industrial applicability.

Claims (14)

Formula (I) below:

(In formula (I), X represents a group containing boron 10 ( ¹⁰ B), L represents a linker, and Y represents an iodine-containing group.) compound or its salt. 2. The compound or salt thereof according to claim 1, wherein ^X in formula (I) is a group containing a boron cluster containing 10B. X in formula (I) is the following formula (XI):

(In formula (XI), a black circle represents BH, a white circle represents B, and * represents a bond with an adjacent atom.). compound or its salt.
Y in formula (I) is the following formula (YI):

(In formula (YI), R ¹ represents a halogen atom or an optionally substituted monovalent organic group, a represents an integer of 1 to 5, and when there are multiple R ^1s , they are each independently and * represents a bond with an adjacent atom, provided that the group represented by formula (YI) contains at least one or more iodine atoms.) The compound or its salt according to any one of claims 1 to 3, characterized by: Y in formula (I) is the following formula (YII):

(In formula (YII), R ² represents an iodine atom, R ³ represents an optionally substituted monovalent alkyl group or an optionally substituted monovalent halogenated alkyl group, b is 1 Represents an integer of ~4, * represents a bond with an adjacent atom.) The compound or salt thereof according to any one of claims 1 to 4, wherein . Y in formula (I) is the following formula (YIII):

(In formula (YIII), R ³ represents an optionally substituted monovalent alkyl group or an optionally substituted monovalent halogenated alkyl group, * represents a bond with an adjacent atom, 6. The compound or salt thereof according to any one of claims 1 to 5, which is a group represented by ). Y in formula (I) is the following formula (YIV):

(In formula (YIV), * represents a bond with an adjacent atom.) The compound or salt thereof according to any one of claims 1 to 6, which is a group represented by . L in formula (I) is the following formula (LI):

(In formula (LI), * represents a bond with an adjacent atom.) The compound or salt thereof according to any one of claims 1 to 7, which is a group represented by . A pharmaceutical composition comprising the compound or salt thereof according to any one of claims 1 to 8. 10. The pharmaceutical composition of Claim 9, further comprising a cell penetrating peptide. 11. The pharmaceutical composition according to claim 10, wherein said cell penetrating peptide is A6K peptide or A6R peptide. The pharmaceutical composition according to any one of claims 9 to 11, which is used for boron neutron capture therapy. A method of treating a tumor disease comprising administering a compound or a salt thereof according to any one of claims 1 to 8 to a patient. administering the compound or salt thereof according to any one of claims 1 to 8 to a patient;
measuring the iodine concentration in the patient's tumor;
estimating the boron concentration in the tumor based on the measured iodine concentration; and
irradiating said patient with neutron beams based on said estimated boron concentration.

Images