Derivatives of 3-borono-phenylalanine

Abstract

To provide a new compound for use in boron neutron capture therapy (BNCT), which has high water solubility and is easy to formulate.SOLUTION: The present invention provides a derivative of 3-borono-phenylalanine represented by the formula (I) or a pharmaceutically acceptable salt thereof. (R1-R4 independently represent H, halogen, a C1-6 alkyl, a C1-6 alkoxy or the like; R5 is H, hydroxy, a C1-6 alkyl, or halogen; R6 is H, or a C1-6 alkyl; R7 is boronic acid (-B(OH)2), boronic acid ester or boronic acid amide).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to derivatives of 3-borono-phenylalanine. [Background technology]

[0002] Boron neutron capture therapy (BNCT) is a cancer treatment method. Boron neutron capture therapy uses the boron-10 isotope ( 10 This is a treatment method in which a boron compound containing B) is taken up by cancer cells, and then low-energy neutron rays (e.g., thermal neutrons) are irradiated to locally destroy the cancer cells through nuclear reactions occurring within the cells. 10 Since selective accumulation of boron compounds containing B in cancer tissue cells is important for improving therapeutic efficacy, it is necessary to develop boron compounds that can be selectively taken up by cancer cells.

[0003] Derivatives of 4-borono-phenylalanine, in which a boron atom or boron atom group has been introduced into the basic skeleton, have been synthesized as drugs for use in BNCT. Drugs currently in clinical use include 4-borono-phenylalanine derivatives (L-BPA) and mercaptoundecahydrododecaborate (BSH). 4-borono-phenylalanine mimics phenylalanine and is taken up by LAT1 (L-type Amino acid Transporter 1), a type of amino acid transporter. However, L-BPA has low water solubility, and formulation requires the addition of solubilizing components such as fructose, making it difficult to formulate (see, for example, Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Mori, Y et.al., (1989) Complex Formation of p-Boronophenylalanine With Some Monosaccharides: Pigment cell research 2, 273-277. Summary of the Invention [Problem to be solved by the invention]

[0005] There is a need to develop new compounds that are highly water-soluble and easy to formulate.

[0006] An object of the present invention is to provide a derivative of 3-borono-phenylalanine. [Means for solving the problem]

[0007] The 4-BPA currently used in BNCT has low water solubility, making it impossible to prepare aqueous formulations without the addition of solubilizers such as fructose or sorbitol. While the addition of such solubilizers makes formulations possible, they can sometimes become unstable, making formulation design difficult.

[0008] The present inventors focused on these problems and conducted extensive research to solve the above problems. As a result, they discovered that a new derivative of 3-borono-phenylalanine has a dramatically increased solubility in water, and thus completed the present invention.

[0009] That is, the present invention provides the following compounds: [1] A compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof: [ka] Here, in formula (I), R 1 , R 2 , R 3 , and R 4are independently H, halogen, hydroxy, cyano, C alkyl, C alkoxy, benzyloxy, C alkoxyC alkyl, nitro, C haloalkyl, aminocarbonyl, C C alkylaminocarbonyl (CONR 8 R 9 (R 8 , R 9 each independently represents H or C1-6 alkyl), C1-C6 alkoxycarbonyl, C1-C6 alkylcarbonyl, COOR 10 (R 10 is H or C1-6 alkyl, amino, alkylamino (NR 11 R 12 (R 11 R 12 each independently represents H or C alkyl), haloalkylsulfanyl, haloalkylsulfinyl, haloalkylsulfonyl, C alkylthio, C alkylsulfinyl, C alkylsulfonyl, aminosulfonyl, sulfo, or sulfamoyl; R 5 represents H, hydroxy, C1-6 alkyl, or halogen; R 6 represents H or C1-6 alkyl; R 7 represents either a boronic acid (-B(OH)2), a boronic acid ester, or a boronic acid amide. (However, R 1 , R 2 , R 3 , and R 4 When all are H, or R 1 , R 2 , R 3 , and R 4 When any one of R is hydroxy, C1-3 alkoxy or methylcarbonyl, R 6 represents C1-6 alkyl) [2] R 6 represents H or methyl, or a pharmaceutically acceptable salt thereof. [3] R 7The compound according to [1] or [2], or a pharmaceutically acceptable salt thereof, wherein represents boronic acid (B(OH)2) or a pinacol ester of boronic acid. [4] R 5 represents H, or a pharmaceutically acceptable salt thereof according to any one of [1] to [3]. [5] R 1 , R 2 , R 3 , and R 4 and any one or more of the following are independently Cl, F, C1-4 alkyl, C1-4 alkoxy, CH2X, CHX2, or CX3 (X represents F), or a pharmaceutically acceptable salt thereof.

[0010] Furthermore, the present invention provides a drug for BNCT, a diagnostic drug, or a drug for injection or infusion for BNCT. [6] A drug for BNCT, comprising the compound according to any one of [1] to [5] or a pharmaceutically acceptable salt thereof. [7] A radioisotope-containing diagnostic agent comprising the compound according to any one of [1] to [5] or a pharmaceutically acceptable salt thereof. [8] A drug for injection and infusion for BNCT, comprising a compound represented by the following formula (I') or a pharmaceutically acceptable salt thereof, and having a total concentration of fructose and sugar alcohol of 0.1% by mass or less: [ka] Here, in formula (I′), R 1 , R 2 , R 3 , and R 4 are independently H, halogen, hydroxy, cyano, C alkyl, C alkoxy, benzyloxy, C alkoxyC alkyl, nitro, C haloalkyl, aminocarbonyl, C C alkylaminocarbonyl (CONR 8 R 9 (R8 , R 9 each independently represents H or C1-6 alkyl), C1-C6 alkoxycarbonyl, C1-C6 alkylcarbonyl, COOR 10 (R 10 is H or C1-6 alkyl, amino, alkylamino (NR 11 R 12 (R 11 R 12 each independently represents H or C alkyl), haloalkylsulfanyl, haloalkylsulfinyl, haloalkylsulfonyl, C alkylthio, C alkylsulfinyl, C alkylsulfonyl, aminosulfonyl, sulfo, or sulfamoyl; R 5 represents H, hydroxy, C1-6 alkyl, or halogen; R 6 represents H or C1-6 alkyl; R 7 represents either a boronic acid (—B(OH)2), a boronic acid ester, or a boronic acid amide. [Effects of the Invention]

[0011] The compound of the present invention or a pharmaceutically acceptable salt thereof is highly water-soluble and can be conveniently used in BNCT and the like. [Brief explanation of the drawings]

[0012] [Figure 1] 1 is a graph showing the results of administering some compounds from the Examples and Comparative Examples to cancer-bearing mice and confirming their distribution in tissues. [Figure 2] 1 is a graph showing the results of administering some other examples of the compounds of the Examples and Comparative Examples to cancer-bearing mice and confirming their distribution in tissues. [Figure 3] 1 is a graph showing the results of administering the compounds of Comparative Example and Example 27 to cancer-bearing mice and confirming their distribution in tissues. DETAILED DESCRIPTION OF THE INVENTION

[0013] In this specification, when a compound having an asymmetric carbon is represented, the compound may be in any of the racemic, R- or S-form unless otherwise specified.

[0014] [3-Borono-phenylalanine derivatives] The 3-borono-phenylalanine derivative of the present invention is a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof: [ka] Here, in formula (I), R 1 , R 2 , R 3 , and R 4 are independently H, halogen, hydroxy, cyano, C alkyl, C alkoxy, benzyloxy, C alkoxyC alkyl, nitro, C haloalkyl, aminocarbonyl, C C alkylaminocarbonyl (CONR 8 R 9 (R 8 , R 9 each independently represents H or C1-6 alkyl), C1-C6 alkoxycarbonyl, C1-C6 alkylcarbonyl, COOR 10 (R 10 is H or C1-6 alkyl, amino, alkylamino (NR 11 R 12 (R 11 R 12 each independently represents H or C alkyl), haloalkylsulfanyl, haloalkylsulfinyl, haloalkylsulfonyl, C alkylthio, C alkylsulfinyl, C alkylsulfonyl, aminosulfonyl, sulfo, or sulfamoyl; R 5 represents H, hydroxy, C1-6 alkyl, or halogen; R 6 represents H or C1-6 alkyl; R 7represents either a boronic acid (-B(OH)2), a boronic acid ester, or a boronic acid amide. (However, R 1 , R 2 , R 3 , and R 4 When all are H, or R 1 , R 2 , R 3 , and R 4 When any one of R is hydroxy, C1-3 alkoxy or methylcarbonyl, R 6 represents C1-6 alkyl)

[0015] In this specification, halogen may be any of F, Cl, Br, and I, but is particularly preferably F, Cl, or Br.

[0016] As used herein, C1-C6 alkyl refers to a linear or branched C1-C6 saturated hydrocarbon group. This definition also includes the use of alkoxy, alkoxyalkyl, and haloalkyl. Examples of C1-6 alkyl groups include methyl, ethyl, propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, t-butyl), pentyl (e.g., n-pentyl, isopentyl, neopentyl), and the like. Preferred are linear or branched C1-C4 alkyl groups, including, but not limited to, methyl, ethyl, isopropyl, and butyl groups.

[0017] As used herein, C1-C6 haloalkyl refers to a C1-C6 alkyl group having one or more halogen substituents. The haloalkyl group is preferably represented by C2X5, CH2X, CHX2, or CX3 (X represents Cl, F, Br, or I), and includes, but is not limited to, for example, CF3, C2F5, CHF2, CCl3, CHCl2, C2Cl5, etc.

[0018] As used herein, C1-C6 alkoxy refers to a group having a linear or branched C1-C6 alkyl group and an oxygen molecule. The C1-C6 alkoxy preferably has a linear or branched C1-C4 alkyl group, including, but not limited to, methoxy, ethoxy, isopropoxy, and butoxy.

[0019] In this specification, C1-6 alkoxyC1-6 alkyl includes, but is not limited to, methoxyethyl, ethoxyethyl and the like.

[0020] As used herein, C1-C6 alkylaminocarbonyl (CONR 8 R 9 (R 8 , R 9 independently represent H or C1-6 alkyl)) includes, but is not limited to, methylaminocarbonyl, dimethylaminocarbonyl, ethylaminocarbonyl, i-propylaminocarbonyl, and the like.

[0021] As used herein, C1-C6 alkylcarbonyl includes, but is not limited to, methylcarbonyl, ethylcarbonyl and the like.

[0022] In this specification, C1-C6 alkoxycarbonyl includes, but is not limited to, methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, isobutoxycarbonyl, sec-butoxycarbonyl, tert-butoxycarbonyl, pentyloxycarbonyl, hexyloxycarbonyl and the like.

[0023] As used herein, COOR 10 (R 10 is H or C1-6 alkyl, amino, alkylamino (NR 11 R 12 (R 11 R 12are each independently H or C1-6 alkyl)) include, but are not limited to, methyloxycarbonyl, aminooxycarbonyl, and the like.

[0024] In this specification, haloalkylsulfanyl, haloalkylsulfinyl, and haloalkylsulfonyl include, but are not limited to, trifluoromethylsulfanyl, trifluoromethylsulfinyl, and trifluoromethylsulfonyl.

[0025] As used herein, C1-C6 alkylthio includes, but is not limited to, methylthio, ethylthio, propylthio, isopropylthio, butylthio, sec-butylthio, tert-butylthio, pentylthio, hexylthio and the like.

[0026] In this specification, C1-C6 alkylsulfinyl includes, but is not limited to, methylsulfinyl, ethylsulfinyl and the like.

[0027] As used herein, C1-C6 alkylsulfonyl includes, but is not limited to, methylsulfonyl, ethylsulfonyl and the like.

[0028] In the derivatives of the present invention, R 7 represents either a boronic acid (—B(OH)2), a boronic acid ester, or a boronic acid amide group, and examples of boronic acid ester or boronic acid amide groups in this definition include R 7 At the position of -B(NR 41 )2, or -B(OR 41 ) 2, or a group having a cyclic structure together with atom B. 41represents a linear or branched C1-C10 alkyl group. Here, the term "linear or branched C1-C10 alkyl group" refers to any alkyl group having 1 to 10 carbon atoms. A linear or branched C1-C8 alkyl group is preferred, and a linear or branched C1-C6 alkyl group is more preferred. Examples of such groups include, but are not limited to, methyl, ethyl, isopropyl, and butyl. Furthermore, the cyclic structure referred to here does not necessarily have only O atoms intervening, but may also have N atoms intervening. Examples include, but are not limited to, groups formed from an ester or ester analogue formed from atom B and any of the group consisting of pinacol, 2,2-dimethyl-1,3-propanediol, N-methyldiethanolamine, 1-6-diaminonaphthalene, N-methyliminodiacetic acid, 1,1,1-trishydroxymethylethane, and catechol. These include, but are not limited to, pinacol boronate esters, MIDA boronate esters, 1,3-propanediol boronate esters, neopentyl glycol boronate esters, catechol boronate esters, pinanediol boronate esters, biscyclohexyldiol boronate esters, MPM boronate esters, trifluoroborate salts, cyclic triol borate salts, and cyclic compounds of diaminonaphthalene amide and boron.

[0029] Of these, R 7 is particularly preferably a boronic acid or a boronic acid ester having a chain or cyclic structure, and most preferably a boronic acid.

[0030] Here, the proportion of boron atoms is not limited, but may be preferably 75% by mass or more, more preferably 80% by mass or more, even more preferably 90% by mass or more, and particularly preferably 95% by mass or more.

[0031] Natural boron (boron) contains boron-10 and boron-11 isotopes, with the boron-10 isotope accounting for 20% and the boron-11 isotope accounting for 80%. Therefore, prior to producing the 3-borono-phenylalanine derivative of the present invention, it is also preferable to enrich boron with a mass number of 10 (boron-10 isotope). In the present invention, for example, commercially available products may be used as the boron atom source. Examples of commercially available products include: 10 B Concentrated boric acid (manufactured by Stella Chemifa Corporation) can be used.

[0032] Here, the boron-10 isotope can be measured by multi-type inductively coupled plasma (ICP) optical emission spectroscopy (ICP-OES) using an Agilent 710 (manufactured by Agilent). The ICP-OES used for the measurement is adjusted in accordance with JIS K0116.

[0033] In the above compounds, the R 1 , R 2 , R 3 , and R 4 It is particularly preferred that any one or more of the following are independently Cl, F, C1-3 alkyl, C1-3 alkoxy, CH2F, CHF2, or CF3.

[0034] In the above compounds, the R 5 It is particularly preferred that R represents H. 1 , R 2 , R 3 , and R 4 It is preferred that not all of are H.

[0035] In the above compounds, the R 6 particularly preferably denotes H, methyl or ethyl.

[0036] In the above compounds, R 7 is particularly preferably a boronic acid (B(OH)2) or a pinacol ester of a boronic acid.

[0037] Among the 3-borono-phenylalanine derivatives of the present invention, one selected from the group consisting of the following compounds or a salt thereof is particularly preferred: (S)-2-Amino-3-(3-borono-4-chlorophenyl)propanoic acid; (S)-2-Amino-3-(3-boronophenyl)-2-methylpropanoic acid; (S)-2-Amino-3-(5-borono-2-fluorophenyl)propanoic acid; (R)-2-amino-3-(5-borono-2-fluorophenyl)propanoic acid; (S)-2-Amino-3-(5-borono-2-chlorophenyl)propanoic acid; (R)-2-amino-3-(5-borono-2-chlorophenyl)propanoic acid; (S)-2-Amino-3-(3-borono-4-fluorophenyl)propanoic acid; (R)-2-amino-3-(3-borono-4-fluorophenyl)propanoic acid; (R)-2-amino-3-(3-borono-4-chlorophenyl)propanoic acid; (S)-2-Amino-3-(5-borono-2-methylphenyl)propanoic acid; (R)-2-amino-3-(5-borono-2-methylphenyl)propanoic acid; (S)-2-Amino-3-(3-borono-4-methoxyphenyl)propanoic acid; (R)-2-amino-3-(3-borono-4-methoxyphenyl)propanoic acid; (R)-2-amino-3-(3-boronophenyl)-2-methylpropanoic acid; (S)-2-Amino-3-(5-borono-2-fluorophenyl)-2-methylpropanoic acid; (R)-2-amino-3-(5-borono-2-fluorophenyl)-2-methylpropanoic acid; (S)-2-Amino-3-(3-borono-4-fluorophenyl)-2-methylpropanoic acid; (R)-2-amino-3-(3-borono-4-fluorophenyl)-2-methylpropanoic acid; (S)-2-Amino-3-(5-borono-2-chlorophenyl)-2-methylpropanoic acid; (R)-2-amino-3-(5-borono-2-chlorophenyl)-2-methylpropanoic acid; (S)-2-Amino-3-(3-borono-4-chlorophenyl)-2-methylpropanoic acid; (R)-2-amino-3-(3-borono-4-chlorophenyl)-2-methylpropanoic acid; (S)-2-Amino-3-(5-borono-2-methylphenyl)-2-methylpropanoic acid; (R)-2-amino-3-(5-borono-2-methylphenyl)-2-methylpropanoic acid; (S)-2-amino-3-(3-borono-4-methoxyphenyl)-2-methylpropanoic acid; and (R)-2-Amino-3-(3-borono-4-methoxyphenyl)-2-methylpropanoic acid.

[0038] In the present invention, "pharmaceutically acceptable salts" include salts with inorganic bases, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, and the like. Suitable examples of salts with inorganic bases include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts; as well as aluminum salts and ammonium salts. Suitable examples of salts with organic bases include salts with trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, N,N'-dibenzylethylenediamine, and the like. Suitable examples of salts with inorganic acids include salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, and the like. Suitable examples of salts with organic acids include salts with formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like. Suitable examples of salts with basic amino acids include salts with arginine, lysine, ornithine, etc., and suitable examples of salts with acidic amino acids include salts with aspartic acid, glutamic acid, etc.

[0039] [Method for producing 3-borono-phenylalanine derivatives] In the present invention, the method for producing the novel 3-borono-phenylalanine derivative is not limited, and a conventional amino acid synthesis method can be used. Although not limited, a particularly preferred method may be, for example, the following method. First, an organic halide represented by the following general formula (II) is reacted with a protected amino acid in the presence of an organic solvent, a basic aqueous solution, and a phase transfer catalyst.

[0040] [ka] Here, in formula (II), R 1 , R 2 , R 3 , and R 4are independently H, halogen, hydroxy, cyano, C alkyl, C alkoxy, benzyloxy, C alkoxyC alkyl, nitro, C haloalkyl, aminocarbonyl, C C alkylaminocarbonyl (CONR 8 R 9 (R 8 , R 9 each independently represents H or C1-6 alkyl), C1-C6 alkoxycarbonyl, C1-C6 alkylcarbonyl, COOR 10 (R 10 is H or C1-6 alkyl, amino, alkylamino (NR 11 R 12 (R 11 R 12 each independently represents H or C alkyl), haloalkylsulfanyl, haloalkylsulfinyl, haloalkylsulfonyl, C alkylthio, C alkylsulfinyl, C alkylsulfonyl, aminosulfonyl, sulfo, or sulfamoyl; R 5 represents H, hydroxy, C1-6 alkyl, or halogen; X 1 , X 2 independently represent halogen, in particular Cl, Br, or I.

[0041] Here, as the organic halide represented by formula (II), a compound prepared by a known method can be used as it is, or a commercially available product can also be used.

[0042] Of these, for example, 2-bromo-4-(bromomethyl)-1-chlorobenzene and 4-bromo-2-(bromomethyl)-1-fluorobenzene are available from Combi-Brocks, 4-bromo-2-(bromomethyl)-1-chlorobenzene is available from BLD Pharmatech Ltd., 2-bromo-4-(bromomethyl)-1-fluorobenzene and 1-(bromomethyl)-3-iodobenzene are available from Tokyo Chemical Industry Co., Ltd., 4-bromo-2-(bromomethyl)-1-methylbenzene is available from Toronto Research Chemicals Inc., and 2-bromo-4-(bromomethyl)-1-methoxybenzene is available from Sigma-Aldrich Japan LLC. Other benzyl bromides are also commercially available. Those that are not commercially available can be prepared, for example, by reacting a correspondingly substituted toluene with N-bromosuccinimide in the presence of 2,2'-azobis(isobutyronitrile). Alternatively, they can be prepared by reducing a correspondingly substituted benzaldehyde or methyl benzoate with sodium borohydride or lithium aluminum hydride to give benzyl alcohol, which can then be brominated with hydrobromic acid or phosphorus tribromide.

[0043] The reaction of the organic halide represented by formula (II) with the protected amino acid can be carried out in an organic solvent, a basic aqueous solution, or in the presence of a phase transfer catalyst. R6 can be H or C1-6 alkyl. Examples of the protected amino acid include p-chlorobenzaldehyde imine and benzophenone imine. Preferably, R6 of the following structure is used: 6 When R is H, it can be a benzophenone imine; 6 When is a methyl group, it can be p-chlorobenzaldehyde imine. [ka]

[0044] The organic solvent used here is not limited, but preferred examples include toluene, benzene, xylene, mesitylene, ethyl ether, isopropyl ether, tetrahydrofuran, dioxane, ethyl acetate, isopropyl acetate, cyclopentyl methyl ether, and methyl t-butyl ether.

[0045] The basic aqueous solution is preferably an aqueous solution of calcium hydroxide, cesium hydroxide, potassium hydroxide, or the like.

[0046] The phase transfer catalyst may be, for example, a Maruoka reagent. The Maruoka reagent is not limited to, but preferably includes, for example, (R)-4,4-dibutyl-2,6-bis(3,4,5-trifluorophenyl)-4,5-dihydro-3H-dinaphtho[2,1-c:1′,2′-e]azepinium bromide, (R)-4,4-dibutyl-2,6-bis(3,4,5-trifluorophenyl)-4,5-dihydro-3H-dinaphtho[2,1-c:1′,2′-e]azepinium bromide, azepinium bromide, (S)-4,4-dibutyl-2,6-bis(3,4,5-trifluorophenyl)-4,5-dihydro-3H-dinaphtho[2,1-c:1′,2′-e]azepinium bromide, (S)-4,4-dibutyl-2,6-bis(3,4,5-trifluorophenyl)-4,5-dihydro-3H-dinaphtho[2,1-c:1′,2′-e]azepinium bromide, and the like can be used.

[0047] The reaction temperature at this time is preferably between −20° C. and 10° C., and the reaction time can be about 1 hour to 60 hours.

[0048] After the reaction is completed, the product can be extracted with an organic solvent such as toluene, and then subjected to washing, drying and filtration steps as appropriate.

[0049] Next, a solvent is added to the reaction product, and the reaction is allowed to proceed with an acid. Preferably, an ether-based solvent is used. Examples of the ether-based solvent include, but are not limited to, diethyl ether, tetrahydrofuran (THF), 2-methyltetrahydrofuran, dioxane, cyclopentyl methyl ether, glyme, and diglyme. In the present invention, tetrahydrofuran is particularly preferably used.

[0050] Examples of the acid include organic acids such as citric acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, trifluoromethanesulfonic acid, toluenesulfonic acid, and methanesulfonic acid, and inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, and phosphoric acid.

[0051] The reaction is carried out at a temperature ranging from 0°C to 50°C.

[0052] The reaction time is about 1 to 10 hours, more preferably 2 to 8 hours, and even more preferably 3 to 6 hours.

[0053] The amino group of the resulting compound is protected by a conventional method. The protecting group is not limited, but for example, a carbamate protecting group, an amide protecting group, or an alkyl protecting group is preferably used. Examples of such carbamate protecting groups include a tert-butoxycarbonyl group (Boc), a benzyloxycarbonyl group (Cbz), a 9-fluorenylmethyloxycarbonyl group (Fmoc), and a 2,2,2-trichloroethoxycarbonyl group (Troc). Examples of amide protecting groups include an acetyl group and a benzoyl group. Examples of alkyl protecting groups include a benzyl group.

[0054] Next, the resulting compound is reacted with a boron compound in a solvent in the presence of a palladium catalyst, an organophosphorus compound and a base.

[0055] Here, examples of the palladium catalyst include, but are not limited to, palladium(II) acetate, palladium(II) chloride, tris(dibenzylideneacetone)dipalladium(0), [1,1′-bis(diphenylphosphine)ferrocene]palladium(II) dichloride dichloromethane adduct, tetrakis(triphenylphosphine)palladium(0), and the like.

[0056] Examples of organophosphorus compounds include, but are not limited to, triphenylphosphine, tricyclohexylphosphine, 1,1′-bis(diphenylphosphino)ferrocene, 2-dicyclohexylphosphino-2′,6′-dimethoxybiphenyl, 2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl, and 2-dicyclohexylphosphino-2′-(N,N-dimethylamino)biphenyl.

[0057] Examples of bases include, but are not limited to, potassium acetate, sodium acetate, sodium carbonate, cesium carbonate, potassium carbonate, and sodium bicarbonate.

[0058] Examples of boron compounds include boric acid esters and boric acid amides, and preferably compounds represented by B(OR)3, B(NR)3, B(OR)2(NR), (RO)2B-B(OR)2, or B(OR)(NR)2 (wherein R is a linear or branched C1-C10 alkyl group, a phenyl group, or a benzyl group). Of these, compounds represented by (RO)2B-B(OR)2 are particularly preferred. The term "linear or branched C1-C10 alkyl group" refers to any alkyl group having 1 to 10 carbon atoms, but is preferably a linear or branched C1-C8 alkyl group, and more preferably a linear or branched C1-C6 alkyl group. Examples of such groups include, but are not limited to, methyl, ethyl, isopropyl, and butyl. Examples of boron compounds include, but are not limited to, bis(pinacolato)diboron.

[0059] Examples of solvents include, but are not limited to, ether solvents such as 1,4-dioxane, tetrahydrofuran, and 1,2-dimethoxyethane; hydrocarbon solvents such as toluene; and polar solvents such as N,N-dimethylformamide and dimethyl sulfoxide. A preferred solvent is dimethyl sulfoxide. The reaction temperature is, for example, 20°C to 160°C, preferably 60°C to 120°C.

[0060] The resulting compounds are then sequentially deprotected to produce the target compound. Deprotection can be carried out in accordance with conventional methods, such as hydrolysis, catalytic hydrogenation, decarboxylation, and oxidation.

[0061] In each step of the production method, purification may be carried out according to a conventional method, which may be modified as appropriate.

[0062] In particular, when the compound is a racemate, it can be used as is, or the optical purity of the R or S isomer can be increased to obtain a compound suitable for use in, for example, boron neutron capture therapy.

[0063] For optical resolution, known techniques may be used as appropriate. For example, a method of optical resolution (using α-chymotrypsin or the like) via a hydrolysis step and an esterification step may be used, as well as a simplified method including a simplified step of using acylase via a hydrolysis step.

[0064] [BNCT (boron neutron capture therapy) drugs] The 3-borono-phenylalanine derivatives of the present invention can be conveniently used for BNCT in the form of the above-mentioned compound or a pharmaceutically acceptable salt thereof, as is, or mixed with a pharmaceutically acceptable carrier in the form of a formulation known to those skilled in the art, or encapsulated in micro / nanoparticles, etc.

[0065] Treatment using a formulation containing a 3-borono-phenylalanine derivative of the present invention is carried out by administering it via any suitable route in such a way that the 3-borono-phenylalanine derivative accumulates in the target tumor. The 3-borono-phenylalanine derivative is preferably concentrated in the tumor before radiation exposure, with a tumor:blood ratio of at least 1.5:1, preferably 2:1 or greater, before radiation exposure. The 3-borono-phenylalanine derivative can be administered all at once or over a continuous period. In some cases, it can also be administered in divided doses. After the compound has accumulated as desired in the tumor, the tumor is irradiated with an effective amount of low-energy neutrons (e.g., epithermal neutrons). The tumor can be irradiated through the skin, or the tumor can be completely or partially exposed before irradiation. Administration of the 3-borono-phenylalanine derivative followed by radiation exposure can be repeated as necessary. If desired, treatment with the 3-borono-phenylalanine derivative can be followed by surgery to shrink the tumor to a surgically feasible size. Alternatively, after surgery, the remaining tumor is destroyed using the 3-borono-phenylalanine derivatives of the present invention. In another embodiment, the patient is administered an appropriate amount of the 3-borono-phenylalanine derivative and irradiated with an effective amount of the naturally occurring neutron emitter, californium-252, which is preferably inserted into the tumor and removed at an appropriate time.

[0066] Here, the type of tumor is not particularly limited, but particularly suitable tumors include brain tumors including glioblastoma and malignant glioma, as well as other head and neck cancers, malignant melanoma, breast cancer, and prostate cancer.Other tumors that may be targeted include epithelial cell cancers such as lung cancer, uterine cancer, kidney cancer, and liver cancer, as well as various sarcomas.

[0067] The 3-borono-phenylalanine derivatives of the present invention can be administered orally or parenterally, for example, intraarterially (e.g., via the carotid artery), intramuscularly, subcutaneously, intramedullary, intrathecally, intraventricularly, intravenously, intraperitoneally, or intranasally.

[0068] The formulations may be in any form, such as powder, granules, fine granules, dry syrup, tablets, capsules, injections, liquids, etc. Depending on the dosage form, the formulation may be mixed with appropriate additives and / or pharmaceutically acceptable carriers by known pharmaceutical techniques and administered to patients alone or in combination with other drugs. Examples of additives include excipients; disintegrants; binders; lubricants; diluents; buffers such as phosphoric acid, citric acid, succinic acid, acetic acid, and other organic acids or their salts; isotonicity agents; preservatives; wetting agents; emulsifiers; dispersants; stabilizers; solubilizers; antioxidants such as ascorbic acid; and low-molecular-weight (less than about 10 residues) polypeptides. Examples of suitable pharmaceutical additives include: (e.g., polyarginine or tripeptides); proteins (e.g., serum albumin, gelatin, or immunoglobulins); hydrophilic polymers (e.g., polyvinylpyrrolidone); amino acids (e.g., glycine, glutamic acid, aspartic acid, or arginine); monosaccharides, disaccharides, and other carbohydrates (including cellulose or its derivatives, glucose, mannose, or dextrin); chelating agents (e.g., EDTA); sugar alcohols (e.g., mannitol or sorbitol); counterions (e.g., sodium); and / or nonionic surfactants (e.g., polysorbate, poloxamer). Pharmaceutical preparations can be prepared by appropriately mixing, diluting, or dissolving these pharmaceutical additives. Preferred carriers include, but are not limited to, pharmaceutically inert aqueous carriers. Examples of such carriers include saline, buffered saline, dextrose, and water. In one embodiment of the present invention, the pharmaceutically acceptable carrier is pharmaceutically inert. Suitable additives and / or pharmaceutically acceptable carriers are non-toxic to recipients at the dosages and concentrations employed. Particularly preferred formulations are injectable solutions prepared with aqueous carriers.

[0069] However, because 3-borono-phenylalanine derivatives are highly soluble in water, when preparing drugs for injection or infusion, it is possible to prepare formulations that are substantially free of solubilizing components such as sugar alcohols (e.g., mannitol or sorbitol) and fructose. For example, it is possible to prepare formulations that contain 0.1% by mass or less, preferably 0.01% by mass or less, and more preferably no monosaccharides and sugar alcohols in total. In particular, it is possible to prepare formulations that contain 0.1% by mass or less, preferably 0.01% by mass or less, and more preferably no fructose and sugar alcohols in total. In such formulations, the 3-borono-phenylalanine derivative may be the following compound or a pharmaceutically acceptable salt thereof:

[0070] [ka] Here, in formula (I), R 1 , R 2 , R 3 , and R 4 are independently H, halogen, hydroxy, cyano, C alkyl, C alkoxy, benzyloxy, C alkoxyC alkyl, nitro, C haloalkyl, aminocarbonyl, C C alkylaminocarbonyl (CONR 8 R 9 (R 8 , R 9 each independently represents H or C1-6 alkyl), C1-C6 alkoxycarbonyl, C1-C6 alkylcarbonyl, COOR 10 (R 10 is H or C1-6 alkyl, amino, alkylamino (NR 11 R 12 (R 11 R 12 each independently represents H or C alkyl), haloalkylsulfanyl, haloalkylsulfinyl, haloalkylsulfonyl, C alkylthio, C alkylsulfinyl, C alkylsulfonyl, aminosulfonyl, sulfo, or sulfamoyl; R 5 represents H, hydroxy, C1-6 alkyl, or halogen; R 6 represents H or C1-6 alkyl; R 7 represents either a boronic acid (—B(OH)2), a boronic acid ester, or a boronic acid amide.

[0071] Techniques for formulation and administration are described, for example, in the latest edition of the Japanese Pharmacopoeia and its latest supplement, "REMINGTON'S PHARMACEUTICAL SCIENCES" (Maack Publishing Co., Easton, PA), last edition.

[0072] The formulations of the 3-borono-phenylalanine derivatives of the present invention contain the desired drug in an amount effective to achieve the intended purpose, and the term "therapeutically effective amount" or "pharmacologically effective amount" is well recognized by those skilled in the art and refers to the amount of drug effective to produce a pharmacological result. The determination of a therapeutically effective dose is well known to those skilled in the art.

[0073] A therapeutically effective amount, as used herein, refers to the amount of drug that alleviates the disease state following administration of radiation. The therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose is preferably within the ED range with little or no toxicity. 50 The circulating concentration of the compound is within a range including the range of 1000 mg / kg / kg of the compound per kg of body weight of the subject to be treated. This dose varies within this range depending on the dosage form used, the patient's sensitivity, and the route of administration. For example, the dosage is appropriately selected depending on the age and other conditions of the patient, the type of disease, the type of conjugate used, and the like. A preferred dosage is, but is not limited to, 5 to 1000 mg / kg of the compound per treatment. In particular, the amount of the compound per kg of body weight of the subject to be treated can be 5 to 500 mg, more preferably 6 to 480 mg.

[0074] [Diagnostic agents containing radioisotopes] The 3-borono-phenylalanine derivative of the present invention can also be prepared as a drug or research reagent containing a radioisotope. When prepared as a drug or research reagent containing a radioisotope, typically, but not limited to, the F atom contained in the compound is 18 The use of F and the use of I atoms in the compound 131 I, 123 I, 124 I, or 125 I, or as a C atom contained in a compound 11 C can be used. The compound obtained in this manner can be used, for example, in RI tests and nuclear medicine tests. These include, but are not limited to, agents for scintigraphic tomography, SPECT (Single Photon Emission Computed Tomography), and PET (Positron Emission Tomography). That is, a radioactive 3-borono-phenylalanine derivative of the present invention is administered to a subject as an agent for PET or SPECT, and images are obtained before treatment to obtain information such as the distribution of the derivative's accumulation in the body and the tumor tissue / normal tissue abundance ratio (T / N ratio). Based on this information, it is also possible to predict the therapeutic effect of BNCT in advance and formulate a treatment plan. The compound thus obtained can also be used as a research reagent. The mode of administration and other details are in accordance with the contents described in the section on BNCT (boron neutron capture therapy) drugs. [Example]

[0075] The present invention will be further described in detail with reference to the following examples, but the invention is not limited thereto.

[0076] In the following examples, the following instruments and reagents were used for analysis and separation and purification of compounds.

[0077] NMR spectrum: (JEOL RESONANCE / JNM-ECZ500R / 500MHz)

[0078] Example 1 [ka] (S)-2-amino-3-(3-borono-4-chlorophenyl)propanoic acid

[0079] Step 1 Preparation of tert-butyl (S)-2-amino-3-(3-bromo-4-chlorophenyl)propanoate tert-Butyl 2-((diphenylmethylene)amino)acetate (6.35 g, 21.5 mmol), 2-bromo-4-(bromomethyl)-1-chlorobenzene (7.36 g, 25.9 mmol), (R)-4,4-dibutyl-2,6-bis(3,4,5-trifluorophenyl)-4,5-dihydro-3H-dinaphtho[2,1-c:1',2'-e]azepinium bromide (16.3 mg, 21.8 μmol) were dissolved in toluene (111 mL) and stirred at -5 °C. Subsequently, 50 w / w% aqueous potassium hydroxide solution (53.56 g) was added dropwise so that the temperature did not exceed 5 °C, and the mixture was stirred at -5 °C for 48 hours. After completion of the reaction, water (30 mL) was added to the reaction solution. The mixture was transferred to a separatory funnel and extracted twice with toluene (50 mL). The organic layer was then washed with saturated brine (50 mL). The organic layer was dried over anhydrous sodium sulfate, the sodium sulfate was removed by filtration, and the resulting organic layer was concentrated under reduced pressure. This concentrate was directly used in the next step without further purification. Tetrahydrofuran (41 mL) was added to the resulting concentrate and stirred at room temperature to dissolve. 25 wt% aqueous citric acid solution (165.30 g) was added and stirred for 7 hours. After completion of the reaction, tetrahydrofuran was distilled off under reduced pressure, and the remaining residue was transferred to a separatory funnel. After washing with ethyl acetate (100 mL), the resulting aqueous layer was adjusted to pH 8 or higher with potassium carbonate. The mixture was transferred to a separatory funnel and extracted twice with ethyl acetate (100 mL). The organic layer was then washed with saturated brine (100 mL). The organic layer was dried over anhydrous sodium sulfate, the sodium sulfate was removed by filtration, and the resulting organic layer was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (n-hexane:ethyl acetate=1:2 (v / v)) to obtain tert-butyl (S)-2-amino-3-(3-bromo-4-chlorophenyl)propanoate (4.55 g, yield 63.3%). 1 H-NMR(CDCl3);1.44(s,9H,t-Bu),2.81(dd,J=7.0,13.5Hz,1H,β-H),2.96(dd,J=5.5,13.5Hz,1H,β-H),3.57(d d,J=6.0,7.5Hz,1H,α-H),7.12(dd,J=2.0,8.0Hz,1H,Ar),7.37(d,J=8.0Hz,1H,Ar),7.50(d,J=2.5Hz,1H,Ar).

[0080] Step 2 Preparation of tert-butyl (S)-3-(3-bromo-4-chlorophenyl)-2-((tert-butoxycarbonyl)amino)propanoate tert-Butyl (S)-2-amino-3-(3-bromo-4-chlorophenyl)propanoate (4.35 g, 13.0 mmol) obtained in Step 1 was dissolved in acetonitrile (44 mL), and BocO (3.41 g, 15.6 mmol) and sodium carbonate (2.76 g, 26.0 mmol) were added. The reaction was allowed to proceed overnight, followed by concentration under reduced pressure. The aqueous layer was then extracted with ethyl acetate (100 mL). The resulting organic layer was dried over anhydrous magnesium sulfate, filtered, and the solvent was then concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography to give the desired product (4.90 g; yield 87%). 1 H-NMR(DMSO-d6);1.34(s,9H,t-Bu),1.36(s,9H,t-Bu),2.84(dd,J=9.5,13.5Hz,1H,β-H),2.96(dd,J=6.0,14.0Hz,1H,β-H),4. 01-4.06(m,1H,α-H),7.20(d,J=8.5Hz,1H,NH),7.28(dd,J=1.5Hz,8.0,Ar),7.53(d,J=8.0Hz,1H,Ar),7.64(d,J=1.5Hz,1H,Ar).

[0081] Step 3 Preparation of (S)-(5-(3-(tert-butoxy)-2-((tert-butoxycarbonyl)amino)-3-oxopropyl)-2-chlorophenyl)boronic acid Pd(PPh3)4 (651 mg, 0.565 mmol), bis(pinacolato)diboron (3.44 g, 13.6 mmol), potassium acetate (2.22 g, 22.6 mmol), and the Boc compound (4.90 g, 11.3 mmol) obtained in Step 2 were added to DMSO (49 mL) and reacted at 80 °C for 18 hours. After completion of the reaction, the reaction mixture was cooled in an ice bath, and ethyl acetate (49 mL) and distilled water (49 mL) were added. After stirring for approximately 5 minutes, the mixture was filtered through Celite, and the filtrate was transferred to a separatory funnel. The aqueous layer was further extracted with ethyl acetate (49 mL). The combined ethyl acetate layers were washed with saturated brine (49 mL). After drying over anhydrous magnesium sulfate, the residue obtained by filtration was crudely purified by silica gel column chromatography.

[0082] The resulting crude product was dissolved in acetone (200 mL). A separate aqueous solution of sodium periodate (4.81 g, 22.5 mmol) and ammonium acetate (1.73 g, 22.5 mmol) dissolved in distilled water (200 mL) was added to this acetone solution. The mixture was then allowed to react at room temperature for 2 days. After the reaction was completed, the acetone was evaporated under reduced pressure, and the resulting aqueous solution was extracted twice with ethyl acetate (100 mL). The extract was then dried over anhydrous magnesium sulfate, filtered, and the ethyl acetate solution was evaporated under reduced pressure. The resulting product was purified by silica gel column chromatography to obtain 2.1 g of the target product (yield: 46%). 1 H-NMR(DMSO-d6);1.34(s,9H,t-Bu),1.37(s,9H,t-Bu),2.80(dd,J=9.5,13.5Hz,1H,β-H),2.91(dd,J=5.5,14.0Hz,1H,β-H),3.94-3.98(m ,1H,α-H),7.12(d,J=8.0Hz,1H,NH),7.19(dd,J=2.5,8.5Hz,Ar),7.24(d,J=9.0Hz,1H,Ar),7.27(d,J=2.0Hz,1H,Ar),8.24(s,2H,B(OH)2).

[0083] Step 4 Preparation of (S)-2-amino-3-(3-borono-4-chlorophenyl)propanoic acid The pinacol-removed product (2.00 g, 5.00 mmol) obtained in Step 3 was dissolved in trifluoroacetic acid (20 mL). After standing for about 3 hours, the solution was concentrated under reduced pressure to obtain 2.2 g of the target product. 1 H-NMR(D2O);3.23(dd,J=7.5,14.5Hz,1H,β-H),3.35(dd,J=5.5,14.0Hz,1H,β-H),4.36(dd,J=6.0,7 .5Hz,1H,α-H),7.31(dd,J=2.0,8.5Hz,1H,Ar),7.41(d,J=2.5Hz,1H,Ar),7.42(d,J=8.0Hz,1H,Ar).

[0084] Example 2 [ka] (S)-2-amino-3-(3-boronophenyl)-2-methylpropanoic acid

[0085] Step 1 Preparation of tert-butyl (S)-2-amino-3-(3-iodophenyl)-2-methylpropanoate tert-Butyl 2-((4-chlorobenzylidene)amino)propanoate (6.68 g, 24.9 mmol), 1-(bromomethyl)-3-iodobenzene (8.92 g, 30.0 mmol), (R)-4,4-dibutyl-2,6-bis(3,4,5-trifluorophenyl)-4,5-dihydro-3H-dinaphtho[2,1-c:1',2'-e]azepinium bromide (19.0 mg, 25.4 μmol) were dissolved in toluene (53 mL) and stirred at -5 °C. Then, 80 w / w% aqueous cesium hydroxide solution (23.42 g) was added dropwise so that the temperature did not exceed 5 °C, and the mixture was stirred at -5 °C for 47 hours. After completion of the reaction, water (30 mL) was added to the reaction solution. The mixture was transferred to a separatory funnel and extracted twice with toluene (50 mL). The organic layer was then washed with saturated brine (50 mL). The organic layer was dried over anhydrous sodium sulfate, the sodium sulfate was removed by filtration, and the resulting organic layer was concentrated under reduced pressure. This concentrate was directly used in the next step without further purification. Tetrahydrofuran (48 mL) was added to the resulting concentrate and stirred at room temperature to dissolve. 25 wt% aqueous citric acid solution (192.09 g) was added and stirred for 7 hours. After completion of the reaction, tetrahydrofuran was distilled off under reduced pressure, and the remaining residue was transferred to a separatory funnel. After washing with ethyl acetate (100 mL), the resulting aqueous layer was adjusted to pH 8 or higher with potassium carbonate. The mixture was transferred to a separatory funnel and extracted twice with ethyl acetate (100 mL). The organic layer was then washed with saturated brine (100 mL). The organic layer was dried over anhydrous sodium sulfate, the sodium sulfate was removed by filtration, and the resulting organic layer was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (n-hexane:ethyl acetate=1:2 (v / v)) to obtain tert-butyl (S)-2-amino-3-(3-bromophenyl)-2-methylpropanoate (5.31 g, yield 58.9%). 1 H-NMR(CDCl3);1.31(s,3H,α-CH3),1.44(s,9H,t-Bu),2.66(d,J=13.0Hz,1H,β-H),3.02(dd,J =13.0Hz,1H,β-H),6.99(t,J=8.0Hz,1H,Ar),7.17(d,J=7.5Hz,1H,Ar),7.53-7.57(m,2H,Ar).

[0086] Step 2 Preparation of tert-butyl (S)-2-((tert-butoxycarbonyl)amino)-3-(3-iodophenyl)-2-methylpropanoate: tert-Butyl (S)-2-amino-3-(3-iodophenyl)-2-methylpropanoate (5.80 g, 16.1 mmol) obtained in Step 1 was dissolved in acetonitrile (58 mL), and BocO (4.22 g, 19.3 mmol) and sodium carbonate (3.41 g, 32.2 mmol) were added. The mixture was allowed to react overnight and then concentrated under reduced pressure. The aqueous layer was extracted with ethyl acetate (120 mL). The resulting organic layer was dried over anhydrous magnesium sulfate and filtered, and the solvent was concentrated under reduced pressure. The resulting residue was solidified with n-hexane and filtered with n-hexane to give the desired product (4.35 g; yield 59%). 1 H-NMR(DMSO-d6);1.11(s,3H,α-CH3),1.38(s,9H,t-Bu),1.43(s,9H,t-Bu),2.80(d,J=13.0Hz,1H,β-H),3.22(d,J=12. 5Hz,1H,β-H),6.87(s,1H,NH),7.08(t,J=7.5Hz,Ar),7.13(d,J=7.0Hz,1H,Ar),7.41(s,1H,Ar),7.57-7.59(m,1H,Ar).

[0087] Step 3 Preparation of (S)-(3-(3-(tert-butoxy)-2-((tert-butoxycarbonyl)amino)-2-methyl-3-oxopropyl)phenyl)boronic acid Pd(dppf)Cl2CH2Cl2 (385 mg, 0.471 mmol), bis(pinacolato)diboron (2.87 g, 11.3 mmol), potassium acetate (1.85 g, 18.8 mmol), and the Boc compound (4.35 g, 9.42 mmol) obtained in Step 2 were added to DMSO (44 mL) and reacted at 100 °C for 2 hours. After completion of the reaction, the reaction mixture was cooled in an ice bath, and ethyl acetate (44 mL) and distilled water (44 mL) were added. After stirring for approximately 5 minutes, the mixture was filtered through Celite, and the filtrate was transferred to a separatory funnel. The aqueous layer was further extracted with ethyl acetate (44 mL). The combined ethyl acetate layers were washed with saturated brine (44 mL). After drying over anhydrous magnesium sulfate, the residue obtained by filtration was crudely purified by silica gel column chromatography.

[0088] The resulting crude product was dissolved in acetone (200 mL). A separate aqueous solution of sodium periodate (4.03 g, 18.9 mmol) and ammonium acetate (1.45 g, 18.9 mmol) dissolved in distilled water (200 mL) was added to the acetone solution. The mixture was then allowed to react at room temperature for two days. After the reaction was completed, the acetone was evaporated under reduced pressure, and the resulting aqueous solution was extracted twice with ethyl acetate (100 mL). The extract was then dried over anhydrous magnesium sulfate, filtered, and the ethyl acetate solution was evaporated under reduced pressure. The resulting product was purified by silica gel column chromatography to obtain 1.9 g of the target product (yield: 53%). 1 H-NMR(DMSO-d6);1.12(s,3H,α-CH3),1.38(s,9H,t-Bu),1.41(s,9H,t-Bu),2.84(d,J=13.0Hz,1H,β-H),3.18(d,J=13.0Hz,1H,β-H ),6.81(s,1H,NH),7.10(d,J=7.0Hz,Ar),7.21(t,J=7.5Hz,1H,Ar),7.51(s,1H,Ar),7.62(d,J=7.5Hz,1H,Ar),7.93(s,2H,B(OH)2).

[0089] Step 4 Preparation of (S)-2-amino-3-(3-boronophenyl)-2-methylpropanoic acid The pinacol-removed product (1.9 g, 5.01 mmol) obtained in Step 3 was dissolved in trifluoroacetic acid (19 mL). After standing for approximately 3 hours, the mixture was concentrated under reduced pressure. The product was dissolved in a small amount of distilled water and neutralized with sodium carbonate, resulting in the precipitation of the target product. This was collected by filtration and washed with cold water to obtain 2.1 g of the target product. 1 H-NMR(D2O);1.76(s,3H,α-CH3),3.19(d,J=14.5Hz,1H,β-H),3.41(d,J=15.0Hz,1H,β-H),7. 42(d,J=7.5Hz,1H,Ar),7.50(t,J=7.5Hz,1H,Ar),7.67(s,1H,Ar),7.81(d,J=7.5Hz,1H,Ar).

[0090] Example 3 The following compound was synthesized in the same manner as in Example 1, except that 2-bromo-4-(bromomethyl)-1-chlorobenzene was changed to 4-bromo-2-(bromomethyl)-1-fluorobenzene. [ka] (S)-2-amino-3-(5-borono-2-fluorophenyl)propanoic acid 1 H-NMR(D2O);3.34(dd,J=7.5,14.5Hz,1H,β-H),3.51(dd,J=6.0,14.5Hz,1H,β-H),4.49(d,J=6.0H z,1H,α-H),7.23(dd,J=8.5,10.5Hz,1H,Ar),7.74(d,J=7.5Hz,1H,Ar),7.80(d,J=6.5Hz,1H,Ar).

[0091] Example 4 The following compound was synthesized in the same manner as in Example 1, except that 2-bromo-4-(bromomethyl)-1-chlorobenzene was changed to 4-bromo-2-(bromomethyl)-1-fluorobenzene and (R)-4,4-dibutyl-2,6-bis(3,4,5-trifluorophenyl)-4,5-dihydro-3H-dinaphtho[2,1-c:1′,2′-e]azepinium bromide was changed to (S)-4,4-dibutyl-2,6-bis(3,4,5-trifluorophenyl)-4,5-dihydro-3H-dinaphtho[2,1-c:1′,2′-e]azepinium bromide. [ka] (R)-2-amino-3-(5-borono-2-fluorophenyl)propanoic acid 1 H-NMR(D2O);3.34(dd,J=8.5,15.0Hz,1H,β-H),3.51(dd,J=5.5,14.5Hz,1H,β-H),4.48(d,J=7.0H z,1H,α-H),7.23(dd,J=8.5,10.0Hz,1H,Ar),7.74(d,J=7.5Hz,1H,Ar),7.80(d,J=6.5Hz,1H,Ar).

[0092] Example 5 The following compound was synthesized in the same manner as in Example 1, except that 2-bromo-4-(bromomethyl)-1-chlorobenzene was changed to 4-bromo-2-(bromomethyl)-1-chlorobenzene. [ka] (S)-2-amino-3-(5-borono-2-chlorophenyl)propanoic acid 1 H-NMR(D2O);3.32(dd,J=8.5,14.0Hz,1H,β-H),3.56(dd,J=6.5,15.0Hz,1H,β-H), 4.48(dd,J=6.0,8.5Hz,1H,α-H),7.47(d,J=8.5Hz,1H,Ar),7.67-7.69(m,2H,Ar).

[0093] Example 6 The following compound was synthesized in the same manner as in Example 1, except that 2-bromo-4-(bromomethyl)-1-chlorobenzene was changed to 4-bromo-2-(bromomethyl)-1-chlorobenzene and (R)-4,4-dibutyl-2,6-bis(3,4,5-trifluorophenyl)-4,5-dihydro-3H-dinaphtho[2,1-c:1′,2′-e]azepinium bromide was changed to (S)-4,4-dibutyl-2,6-bis(3,4,5-trifluorophenyl)-4,5-dihydro-3H-dinaphtho[2,1-c:1′,2′-e]azepinium bromide.

[0094] [ka] (R)-2-amino-3-(5-borono-2-chlorophenyl)propanoic acid 1 H-NMR(D2O);3.32(dd,J=8.5,14.5Hz,1H,β-H),3.56(dd,J=6.0,14.0Hz,1H,β-H), 4.49(dd,J=6.0,8.5Hz,1H,α-H),7.47(d,J=9.0Hz,1H,Ar),7.67-7.69(m,2H,Ar).

[0095] Example 7 The following compound was synthesized in the same manner as in Example 1, except that 2-bromo-4-(bromomethyl)-1-chlorobenzene was changed to 2-bromo-4-(bromomethyl)-1-fluorobenzene. [ka] (S)-2-amino-3-(3-borono-4-fluorophenyl)propanoic acid 1 H-NMR(D2O);3.25(dd,J=7.0,14.0Hz,1H,β-H),3.38(dd,J=5.0,14.0Hz,1H,β-H),4.40(dd,J=6.0 ,8.0Hz,1H,α-H),7.14(t,J=9.0Hz,1H,Ar),7.41-7.45(m,1H,Ar),7.55(dd,J=2.0,5.5Hz,1H,Ar).

[0096] Example 8 The following compound was synthesized in the same manner as in Example 1, except that 2-bromo-4-(bromomethyl)-1-chlorobenzene was replaced with 2-bromo-4-(bromomethyl)-1-fluorobenzene, and (R)-4,4-dibutyl-2,6-bis(3,4,5-trifluorophenyl)-4,5-dihydro-3H-dinaphtho[2,1-c:1′,2′-e]azepinium bromide was replaced with (S)-4,4-dibutyl-2,6-bis(3,4,5-trifluorophenyl)-4,5-dihydro-3H-dinaphtho[2,1-c:1′,2′-e]azepinium bromide. [ka] (R)-2-amino-3-(3-borono-4-fluorophenyl)propanoic acid 1 H-NMR(D2O);3.25(dd,J=7.0,14.0Hz,1H,β-H),3.38(dd,J=5.0,14.0Hz,1H,β-H),4.40(dd,J=6.0 ,8.0Hz,1H,α-H),7.14(t,J=9.0Hz,1H,Ar),7.41-7.45(m,1H,Ar),7.55(dd,J=2.0,5.5Hz,1H,Ar).

[0097] Example 9 The following compound was synthesized in the same manner as in Example 1, except that (R)-4,4-dibutyl-2,6-bis(3,4,5-trifluorophenyl)-4,5-dihydro-3H-dinaphtho[2,1-c:1′,2′-e]azepinium bromide was changed to (S)-4,4-dibutyl-2,6-bis(3,4,5-trifluorophenyl)-4,5-dihydro-3H-dinaphtho[2,1-c:1′,2′-e]azepinium bromide. [ka] (R)-2-amino-3-(3-borono-4-chlorophenyl)propanoic acid 1H-NMR(D2O);3.23(dd,J=8.0,15.0Hz,1H,β-H),3.35(dd,J=5.5,14.0Hz,1H,β-H),4.36(dd,J=6.0,7 .5Hz,1H,α-H),7.31(dd,J=2.0,8.5Hz,1H,Ar),7.41(d,J=2.5Hz,1H,Ar),7.42(d,J=8.0Hz,1H,Ar).

[0098] Example 10 The following compound was synthesized in the same manner as in Example 1, except that 2-bromo-4-(bromomethyl)-1-chlorobenzene was changed to 4-bromo-2-(bromomethyl)-1-methylbenzene. [ka] (S)-2-amino-3-(5-borono-2-methylphenyl)propanoic acid 1 H-NMR(D2O);3.23(dd,J=8.0,15.0Hz,1H,β-H),3.35(dd,J=5.5,14.0Hz,1H,β-H),4.36(dd,J=6.0,7 .5Hz,1H,α-H),7.31(dd,J=2.0,8.5Hz,1H,Ar),7.41(d,J=2.5Hz,1H,Ar),7.42(d,J=8.0Hz,1H,Ar).

[0099] Example 11 The following compound was synthesized in the same manner as in Example 1, except that 2-bromo-4-(bromomethyl)-1-chlorobenzene was changed to 4-bromo-2-(bromomethyl)-1-methylbenzene, and (R)-4,4-dibutyl-2,6-bis(3,4,5-trifluorophenyl)-4,5-dihydro-3H-dinaphtho[2,1-c:1′,2′-e]azepinium bromide was changed to (S)-4,4-dibutyl-2,6-bis(3,4,5-trifluorophenyl)-4,5-dihydro-3H-dinaphtho[2,1-c:1′,2′-e]azepinium bromide. [ka] (R)-2-amino-3-(5-borono-2-methylphenyl)propanoic acid 1 H-NMR(D2O);3.23(dd,J=8.0,15.0Hz,1H,β-H),3.35(dd,J=5.5,14.0Hz,1H,β-H),4.36(dd,J=6.0,7 .5Hz,1H,α-H),7.31(dd,J=2.0,8.5Hz,1H,Ar),7.41(d,J=2.5Hz,1H,Ar),7.42(d,J=8.0Hz,1H,Ar).

[0100] Example 12 The following compound was synthesized in the same manner as in Example 1, except that 2-bromo-4-(bromomethyl)-1-chlorobenzene was changed to 2-bromo-4-(bromomethyl)-1-methoxybenzene. [ka] (S)-2-amino-3-(3-borono-4-methoxyphenyl)propanoic acid 1 H-NMR(D2O);3.23(dd,J=8.0,15.0Hz,1H,β-H),3.35(dd,J=5.5,14.0Hz,1H,β-H),4.36(dd,J=6.0,7 .5Hz,1H,α-H),7.31(dd,J=2.0,8.5Hz,1H,Ar),7.41(d,J=2.5Hz,1H,Ar),7.42(d,J=8.0Hz,1H,Ar).

[0101] Example 13 The following compound was synthesized in the same manner as in Example 1, except that 2-bromo-4-(bromomethyl)-1-chlorobenzene was replaced with 2-bromo-4-(bromomethyl)-1-methoxybenzene, and (R)-4,4-dibutyl-2,6-bis(3,4,5-trifluorophenyl)-4,5-dihydro-3H-dinaphtho[2,1-c:1′,2′-e]azepinium bromide was replaced with (S)-4,4-dibutyl-2,6-bis(3,4,5-trifluorophenyl)-4,5-dihydro-3H-dinaphtho[2,1-c:1′,2′-e]azepinium bromide. [ka] (R)-2-amino-3-(3-borono-4-methoxyphenyl)propanoic acid 1 H-NMR(D2O);3.23(dd,J=8.0,15.0Hz,1H,β-H),3.35(dd,J=5.5,14.0Hz,1H,β-H),4.36(dd,J=6.0,7 .5Hz,1H,α-H),7.31(dd,J=2.0,8.5Hz,1H,Ar),7.41(d,J=2.5Hz,1H,Ar),7.42(d,J=8.0Hz,1H,Ar).

[0102] Example 14 The following compound was synthesized in the same manner as in Example 2, except that (R)-4,4-dibutyl-2,6-bis(3,4,5-trifluorophenyl)-4,5-dihydro-3H-dinaphtho[2,1-c:1′,2′-e]azepinium bromide was replaced with (S)-4,4-dibutyl-2,6-bis(3,4,5-trifluorophenyl)-4,5-dihydro-3H-dinaphtho[2,1-c:1′,2′-e]azepinium bromide. [ka] (R)-2-amino-3-(3-boronophenyl)-2-methylpropanoic acid 1 H-NMR(D2O);1.67(s,3H,α-CH3),3.16(d,J=14.5Hz,1H,β-H),3.39(d,J=14.5Hz,1H,β-H),7. 38(d,J=8.0Hz,1H,Ar),7.46(t,J=7.5Hz,1H,Ar),7.60(s,1H,Ar),7.74(d,J=7.5Hz,1H,Ar).

[0103] Example 15 The following compound was synthesized in the same manner as in Example 2, except that 1-(bromomethyl)-3-iodobenzene was changed to 4-bromo-2-(bromomethyl)-1-fluorobenzene. [ka] (S)-2-amino-3-(5-borono-2-fluorophenyl)-2-methylpropanoic acid 1 H-NMR(D2O);1.67(s,3H,α-CH3),3.31(s,2H,β-H),7.20(dd,J=8.0,10.5Hz,1H,Ar),7.63(dd,J=1.5,8.0Hz,1H,Ar),7.73-7.77(m,1H,Ar).

[0104] Example 16 The following compound was synthesized in the same manner as in Example 2, except that 1-(bromomethyl)-3-iodobenzene was changed to 4-bromo-2-(bromomethyl)-1-fluorobenzene and (R)-4,4-dibutyl-2,6-bis(3,4,5-trifluorophenyl)-4,5-dihydro-3H-dinaphtho[2,1-c:1′,2′-e]azepinium bromide was changed to (S)-4,4-dibutyl-2,6-bis(3,4,5-trifluorophenyl)-4,5-dihydro-3H-dinaphtho[2,1-c:1′,2′-e]azepinium bromide. [ka] (R)-2-amino-3-(5-borono-2-fluorophenyl)-2-methylpropanoic acid 1 H-NMR(D2O);1.77(s,3H,α-CH3),3.33(s,2H,β-H),7.24(dd,J=8.5,10.0Hz,1H,Ar),7.71(dd,J=1.5,8.0Hz,1H,Ar),7.81-7.84(m,1H,Ar).

[0105] Example 17 The following compound was synthesized in the same manner as in Example 2, except that 1-(bromomethyl)-3-iodobenzene was changed to 2-bromo-4-(bromomethyl)-1-fluorobenzene. [ka] (S)-2-amino-3-(3-borono-4-fluorophenyl)-2-methylpropanoic acid 1 H-NMR(D2O);1.74(s,3H,α-CH3),3.19(d,J=14.0Hz,1H,β-H),3.39(d,J=14.0Hz,1 H,β-H),7.15(t,J=8.5Hz,1H,Ar),7.39(m,1H,Ar),7.53(dd,J=2.0,5.5Hz,1H,Ar).

[0106] Example 18 The following compound was synthesized in the same manner as in Example 2, except that 1-(bromomethyl)-3-iodobenzene was changed to 2-bromo-4-(bromomethyl)-1-fluorobenzene and (R)-4,4-dibutyl-2,6-bis(3,4,5-trifluorophenyl)-4,5-dihydro-3H-dinaphtho[2,1-c:1′,2′-e]azepinium bromide was changed to (S)-4,4-dibutyl-2,6-bis(3,4,5-trifluorophenyl)-4,5-dihydro-3H-dinaphtho[2,1-c:1′,2′-e]azepinium bromide. [ka] (R)-2-amino-3-(3-borono-4-fluorophenyl)-2-methylpropanoic acid 1 H-NMR(D2O);1.74(s,3H,α-CH3),3.19(d,J=14.0Hz,1H,β-H),3.39(d,J=14.0Hz,1 H,β-H),7.15(t,J=8.5Hz,1H,Ar),7.39(m,1H,Ar),7.53(dd,J=2.0,5.5Hz,1H,Ar).

[0107] Example 19 The following compound was synthesized in the same manner as in Example 2, except that 1-(bromomethyl)-3-iodobenzene was changed to 4-bromo-2-(bromomethyl)-1-chlorobenzene. [ka] (S)-2-amino-3-(5-borono-2-chlorophenyl)-2-methylpropanoic acid 1H-NMR(D2O);1.73(s,3H,α-CH3),3.38(d,J=14.5Hz,1H,β-H),3.50(d,J=15.0Hz,1H,β-H),7.48(d,J=8.0Hz,1H,Ar),7.65-7.68(m,1H,Ar).

[0108] Example 20 The following compound was synthesized in the same manner as in Example 2, except that 1-(bromomethyl)-3-iodobenzene was changed to 4-bromo-2-(bromomethyl)-1-chlorobenzene and (R)-4,4-dibutyl-2,6-bis(3,4,5-trifluorophenyl)-4,5-dihydro-3H-dinaphtho[2,1-c:1′,2′-e]azepinium bromide was changed to (S)-4,4-dibutyl-2,6-bis(3,4,5-trifluorophenyl)-4,5-dihydro-3H-dinaphtho[2,1-c:1′,2′-e]azepinium bromide. [ka] (R)-2-amino-3-(5-borono-2-chlorophenyl)-2-methylpropanoic acid 1 H-NMR(D2O);1.77(s,3H,α-CH3),3.42(d,J=14.5Hz,1H,β-H),3.54(d,J=15.0Hz,1H,β-H),7.49(d,J=8.5Hz,1H,Ar),7.71-7.72(m,1H,Ar).

[0109] Example 21 The following compound was synthesized in the same manner as in Example 2, except that 1-(bromomethyl)-3-iodobenzene was changed to 2-bromo-4-(bromomethyl)-1-chlorobenzene. [ka] (S)-2-amino-3-(3-borono-4-chlorophenyl)-2-methylpropanoic acid 1H-NMR(D2O);1.74(s,3H,α-CH3),3.19(d,J=14.0Hz,1H,β-H),3.39(d,J=14.0Hz,1 H,β-H),7.15(t,J=8.5Hz,1H,Ar),7.39(m,1H,Ar),7.53(dd,J=2.0,5.5Hz,1H,Ar).

[0110] Example 22 The following compound was synthesized in the same manner as in Example 2, except that 1-(bromomethyl)-3-iodobenzene was changed to 2-bromo-4-(bromomethyl)-1-chlorobenzene and (R)-4,4-dibutyl-2,6-bis(3,4,5-trifluorophenyl)-4,5-dihydro-3H-dinaphtho[2,1-c:1′,2′-e]azepinium bromide was changed to (S)-4,4-dibutyl-2,6-bis(3,4,5-trifluorophenyl)-4,5-dihydro-3H-dinaphtho[2,1-c:1′,2′-e]azepinium bromide. [ka] (R)-2-amino-3-(3-borono-4-chlorophenyl)-2-methylpropanoic acid 1 H-NMR(D2O);1.74(s,3H,α-CH3),3.19(d,J=14.0Hz,1H,β-H),3.39(d,J=14.0Hz,1 H,β-H),7.15(t,J=8.5Hz,1H,Ar),7.39(m,1H,Ar),7.53(dd,J=2.0,5.5Hz,1H,Ar).

[0111] Example 23 The following compound was synthesized in the same manner as in Example 2, except that 1-(bromomethyl)-3-iodobenzene was changed to 4-bromo-2-(bromomethyl)-1-methylbenzene. [ka] (S)-2-amino-3-(5-borono-2-methylphenyl)-2-methylpropanoic acid 1H-NMR(D2O);3.23(dd,J=8.0,15.0Hz,1H,β-H),3.35(dd,J=5.5,14.0Hz,1H,β-H),4.36(dd,J=6.0,7 .5Hz,1H,α-H),7.31(dd,J=2.0,8.5Hz,1H,Ar),7.41(d,J=2.5Hz,1H,Ar),7.42(d,J=8.0Hz,1H,Ar).

[0112] Example 24 The following compound was synthesized in the same manner as in Example 2, except that 1-(bromomethyl)-3-iodobenzene was changed to 4-bromo-2-(bromomethyl)-1-methylbenzene and (R)-4,4-dibutyl-2,6-bis(3,4,5-trifluorophenyl)-4,5-dihydro-3H-dinaphtho[2,1-c:1′,2′-e]azepinium bromide was changed to (S)-4,4-dibutyl-2,6-bis(3,4,5-trifluorophenyl)-4,5-dihydro-3H-dinaphtho[2,1-c:1′,2′-e]azepinium bromide. [ka] (R)-2-amino-3-(5-borono-2-methylphenyl)-2-methylpropanoic acid 1 H-NMR(D2O);3.23(dd,J=8.0,15.0Hz,1H,β-H),3.35(dd,J=5.5,14.0Hz,1H,β-H),4.36(dd,J=6.0,7 .5Hz,1H,α-H),7.31(dd,J=2.0,8.5Hz,1H,Ar),7.41(d,J=2.5Hz,1H,Ar),7.42(d,J=8.0Hz,1H,Ar).

[0113] Example 25 The following compound was synthesized in the same manner as in Example 2, except that 1-(bromomethyl)-3-iodobenzene was changed to 2-bromo-4-(bromomethyl)-1-methoxybenzene. [ka] (S)-2-amino-3-(3-borono-4-methoxyphenyl)-2-methylpropanoic acid 1 H-NMR(D2O);3.23(dd,J=8.0,15.0Hz,1H,β-H),3.35(dd,J=5.5,14.0Hz,1H,β-H),4.36(dd,J=6.0,7 .5Hz,1H,α-H),7.31(dd,J=2.0,8.5Hz,1H,Ar),7.41(d,J=2.5Hz,1H,Ar),7.42(d,J=8.0Hz,1H,Ar).

[0114] Example 26 The following compound was synthesized in the same manner as in Example 2, except that 1-(bromomethyl)-3-iodobenzene was changed to 2-bromo-4-(bromomethyl)-1-methoxybenzene and (R)-4,4-dibutyl-2,6-bis(3,4,5-trifluorophenyl)-4,5-dihydro-3H-dinaphtho[2,1-c:1′,2′-e]azepinium bromide was changed to (S)-4,4-dibutyl-2,6-bis(3,4,5-trifluorophenyl)-4,5-dihydro-3H-dinaphtho[2,1-c:1′,2′-e]azepinium bromide. [ka] (R)-2-amino-3-(3-borono-4-methoxyphenyl)-2-methylpropanoic acid 1 H-NMR(D2O);3.23(dd,J=8.0,15.0Hz,1H,β-H),3.35(dd,J=5.5,14.0Hz,1H,β-H),4.36(dd,J=6.0,7 .5Hz,1H,α-H),7.31(dd,J=2.0,8.5Hz,1H,Ar),7.41(d,J=2.5Hz,1H,Ar),7.42(d,J=8.0Hz,1H,Ar).

[0115] Example 27 (S)-2-amino-3-(3-boronophenyl)propanoic acid was synthesized in the same manner as in Example 1, except that 1-(bromomethyl)-3-iodobenzene was changed to 1-(bromomethyl)-3-iodobenzene.

[0116] (Comparative Example 1) 4-boronophenylalanine ( 10 B concentrate) was used.

[0117] [Uptake test] The compounds obtained in the Examples and Comparative Examples were evaluated for uptake using cultured cells. Three types of cells were used: SAS (human tongue squamous cell carcinoma), MCF-7 (human breast cancer), and A172 (human glioma). L-BPA was used as a control compound, and the test was carried out three times for each compound obtained in each example.

[0118] Prepare cells to reach logarithmic growth phase on the day of seeding, and soak them in 5 x 10 6 The cells were seeded onto 100 mm dishes at a seeding density of 10 cells / dish and pre-cultured at 37°C in a 5% CO2 atmosphere for 24 hours.

[0119] After pre-culture, the medium was replaced with 7 mL of medium containing the control compound and each compound of the example at 1 mM, and the cells were exposed to the compounds at 37°C in a 5% CO 2 atmosphere for 3 hours. After exposure to the compounds, the cells were harvested with trypsin and counted. After packing the cells by centrifugation, 0.6 mL of hydrogen peroxide and 0.3 mL of perchloric acid were added to the cells and ashed overnight at 75°C.

[0120] The ashed sample was made up to 5 mL with water and filtered through No. 5C filter paper to obtain the filtrate used for measuring the boron concentration. The boron concentration of the sample was measured using an Agilent 710 ICP-OES with boric acid standard solution as the standard. The amount of uptake of the compound of the example in each cell was measured at 10 cells / mL. 7 The amount of boron (μg) per cell was calculated, and the uptake ratio to L-BPA was calculated from the results of the simultaneous uptake of L-BPA. As a result, it was found that the evaluation using the compounds of the examples showed an uptake amount almost equivalent to that of L-BPA.

[0121] [Evaluation of water solubility] An appropriate amount of each compound of the examples was measured and dissolved in water. 1M aqueous sodium hydroxide solution was added dropwise, and the solution was neutralized while checking with pH test paper. The prepared agent was analyzed by ICP to calculate the boron concentration, which was used as the sample concentration.

[0122] The results of the water solubility evaluation test of representative example compounds are shown in Table 1. The values ​​in Table 1 are values ​​calculated from the average of three measurements. [Table 1]

[0123] This result demonstrates that the compounds of the examples have extremely high solubility in water compared to the compounds of the comparative examples.

[0124] [Uptake test] 1. Construction of an evaluation system for selective uptake of LAT1 and LAT2 (1) Construction of HEK293 cell lines overexpressing human LAT1 and LAT2 HEK293 cells stably expressing high levels of human LAT1 and LAT2 were prepared according to the method described in Khunweeraphong, N. et al. (Journal of Pharmacology Science, 2012, vol. 119, pp. 368-380). Shuttle vector DNA (LAT1: EX-H4509-M02, LAT2: EX-U0514-M02, GeneCopoeia) was constructed containing ampicillin and neomycin resistance markers and the full-length cDNA of human LAT1 or LAT2 inserted under the CMV (cytomegalovirus) promoter. These vectors were then transfected into HEK293 cells using Lipofectamine® 2000 (Invitrogen) according to the manufacturer's instructions. Then, cell clones stably expressing the introduced gene were selected by limiting dilution in the presence of 0.9 mg / mL Geneticin (registered trademark), and cell clones showing increased L-boronophenylalanine (L-BPA) uptake by approximately 2 to 5 times compared to the HEK293 cells before gene introduction were obtained. These cells were then passaged and used to evaluate the selective uptake of LAT1 and LAT2.

[0125] (2) Evaluation of selective uptake of LAT1 and LAT2 The method for evaluating cellular uptake was the same as that described in Khunweeraphong, N et al. (Journal of Pharmacology Science, 2012, vol. 119, pp. 368-380). The cells obtained in (1) were used for the evaluation. However, radioisotopes were not used. Instead, a substrate concentration of 0.1 mM was used, and 2 mM BCH (2-amino-2-norbornanecarboxylic acid) was used as the LAT1 and LAT2 inhibitor. After the reaction, the cells were recovered with 0.05% Tween 20, and the concentration of 4-borono-phenylalanine derivatives in the resulting cell solution was determined. Quantification of intracellular 4-borono-phenylalanine derivatives was performed according to the method described in Hattori, Y et al. (Sensors 2017, 17, 2436), using 2-(2-hydroxyphenyl)pyridine (boron sensor 5) as the boron sensor. To account for inter-experimental variability, cellular uptake was evaluated as a relative value (LAT1 selectivity) to the uptake of L-BPA, a control performed on the same day. LAT1 selectivity = (quantitative uptake of compound in LAT1 cells / quantitative uptake of control L-BPA in LAT1 cells) / (quantitative uptake of compound in LAT2 cells / quantitative uptake of control L-BPA in LAT2 cells), as follows:

number

[0126] [Single toxicity test] Place 17.6 mL of 1 mol / L NaOH into a beaker, then add 24 mL of water for injection and mix. Add 2.52 g of D-sorbitol. After confirming complete dissolution, add 17.1 mL of 1 mol / L hydrochloric acid to adjust the pH to 7.4-7.8. Transfer to a measuring cylinder, rinse the beaker with water for injection, and transfer the rinse solution to the measuring cylinder. Repeat this process as many times as possible, rinsing the beaker while bringing the total volume to 80 mL. Sterilize by filtration using a filter (Millex GV, pore size 0.22 μm, Merck, sterile disposable product) to make a 3.15% D-sorbitol solution.

[0127] Separately, place 10.1 mL of 1 mol / L NaOH in a beaker, add 13.8 mL of water for injection, and mix. Add 2.30 g of the test substance and 1.45 g of D-sorbitol. After confirming complete dissolution, add 1.36 mL of 1 mol / L hydrochloric acid to adjust the pH to 7.4-7.8. Transfer to a measuring cylinder, rinse the beaker with water for injection, and transfer the rinse solution to the measuring cylinder. Repeat this process as many times as possible, rinsing the beaker while bringing the total volume to 46 mL. Sterilize by filtration using a filter (Millex GV, pore size 0.22 μm, Merck, sterile disposable product) to make a 50 mg / mL solution.

[0128] The animals were placed in a restrainer (Ballman cage), and the administration solution was filled with a 10 mL polypropylene syringe and a 24-gauge indwelling needle (Surflow F&F, Terumo Corporation, both sterilized disposable products) and injected into the tail vein at a rate of 1 mL / kg / min using a microprocessor single syringe pump (Pump11 Elite, Harvard Apparatus Inc.). The doses were 250, 500, and 1000 mg / kg, with five animals per group. After administration was completed, the animals were returned to their cages.

[0129] The observation period will be 8 days, including the day of administration. The day of administration will be designated Day 1, the day after administration will be designated Day 2, and subsequent days will be indicated. Observation frequency will be 6 times on the day of administration (just before administration, and 5, 30, 60 minutes, 2, and 4 hours after administration), and once a day from the day after administration. Individual observations will be made from outside the cage, and animals suspected of showing abnormalities will be removed from their cages and observed. Body weight will be measured on Day 1 (before administration), Day 4, and Day 8.

[0130] [Distribution test using tumor-bearing mice 1] Human pancreatic cancer T3M-4 cells were cultured in HAM-F12 medium. The cells were detached with trypsin solution and collected by centrifugation. 4x10 6Cells were suspended in PBS(-) at a concentration of 100 cells / 100 μL. The cell suspension was subcutaneously injected (100 μL / mouse) into the right lower leg of BALB / c nu / nu mice (male, 4 weeks old) using a 26G needle. The size of the tumors formed was then visually observed for 3-4 weeks, and tumors reaching approximately 4-10 mm in size were used for distribution experiments.

[0131] Separately, formulations for injection into mice were prepared. The compounds of Comparative Example 1 and the Examples were prepared as injections. Comparative Example 1 was insoluble in water and could not be formulated as is. Therefore, the compound of Comparative Example 1 and fructose were dissolved in aqueous sodium hydroxide, neutralized with hydrochloric acid, and then mixed with saline or PBS(-). An injection was prepared so that the final compound concentration was 1% by mass and fructose 2.2% by mass. On the other hand, the compounds of Examples 2 and 27 could be directly dissolved in saline or PBS(-) to prepare an injection. 100 μL of each compound was administered via the tail vein to tumor-bearing mice, with a target concentration of 10 mg / mL. Sixty minutes after administration, the mice were sacrificed, dissected, and organ weights were measured. The excised organs were ashed with nitric acid, and the boron content was quantified using ICP-MS or ICP-OES.

[0132] The results of the distribution test of the compounds of Comparative Example 1, Example 2, and Example 27 are shown in Figure 1. As the distribution in the subcutaneously transplanted tumor, plasma, muscle, or skin, the boron accumulation rate per unit mass (%ID / g) was expressed as the mean ± standard deviation of four animals.

[0133] Similarly, the results of the distribution test for the compounds of Examples 7, 15, and 17 and Example 27 are shown in Figure 2. As distribution to tumor, plasma, muscle, or skin, the boron accumulation rate per unit mass (%ID / g) is expressed as the mean ± standard deviation of four animals.

[0134] [Distribution test using tumor-bearing mice 2] The compound of Example 27 was prepared as an injection in the presence or absence of fructose. Specifically, the compound of Example 27 and fructose were dissolved in aqueous sodium hydroxide, neutralized with hydrochloric acid, and then mixed with saline or PBS(-) to prepare an injection so that the final concentration of the compound was 1% by mass and fructose was 2.2% by mass. On the other hand, in the absence of fructose, the compound was directly dissolved in saline or PBS(-) to prepare an injection. As a result, the compound of Example 27 could be formulated under all conditions. Using this fructose-containing formulation, the fructose-free formulation, and the fructose-containing formulation containing the compound of Comparative Example 1, 100 μL of each compound was administered to tumor-bearing mice via the tail vein in the same manner as in [Distribution Test 1 Using Cancer-Bearing Mice], with the aim of achieving a concentration of 10 mg / mL. Sixty minutes after administration, the mice were sacrificed, dissected, and their organ weights were measured. The excised organs were incinerated with nitric acid, and the boron content was quantified using ICP-MS or ICP-OES.

[0135] As a result, it was found that the compound of Example 27, whether containing fructose or not, showed the same distribution state. Furthermore, the amount of boron accumulated in the tumor was the same for the compounds of Comparative Example 1 and Example 27.

[0136] [Distribution ratio within tumor-bearing mice] Next, from these results, the amount of boron accumulated in the tumor relative to the amount of boron accumulated in normal tissues, muscle and plasma, was calculated as a ratio, and the ratio is shown in Table 2 below. These results demonstrate that the compounds of the Examples have tumor accumulation properties equal to or greater than that of the compounds of the Comparative Examples. [Table 2]

[0137] [Km value measurement] The Km was evaluated using the cellular uptake method described by Khunweeraphong, N. et al. (Journal of Pharmacology Science, 2012, Vol. 119, pp. 368-380), with an uptake time of 2-3 minutes. However, radioisotopes were not used. Cells were incubated with multiple substrate concentrations, recovered with 0.05% Tween 20, and the concentration of the boron compound in the resulting cell solution was determined. HEK293 cells stably expressing the human LAT1 or human LAT2 transporter were used, established according to the method described in (Journal of Pharmacology Science, 2012, Vol. 119, pp. 368-380). Intracellular boron compounds were quantified using the method described by Hattori et al. (Sensors 2017, 17, 2436) using 2-(2-hydroxyphenyl)pyridine (boron sensor 5) as the boron sensor. Km was calculated using Lineweaver-Burk analysis.

[0138] [Efflux measurement] LAT1-expressing HEK293 cells were used to measure the amount of boron compounds taken up into the cells after substrate uptake and the effluent was added. The substrate concentration was 0.1 mM, and the LAT1 effluent was HBSS (Na+-free) and 0.05 mM leucine. After the reaction, the cells were recovered with 0.05% Tween 20, and the boron compound concentration in the resulting cell solution was determined. The effluent time was 1-10 minutes. Quantification of intracellular boron compounds was performed according to the method described in Hattori, Y. et al. (Sensors 2017, 17, 2436), using 2-(2-hydroxyphenyl)pyridine (boron sensor 5) as the boron sensor. The cellular uptake is evaluated as a relative value to the cell fluid before outflow, which serves as a comparison.

[0139] [Metabolic stability test] Using commercially available pooled human liver microsomes, the target compound is reacted for a certain period of time, and the residual rate is calculated by comparing the reacted sample with the unreacted sample, thereby evaluating the degree of metabolism in the liver.

[0140] Human liver microsomes (0.5 mg protein / mL) were incubated in 0.2 mL of buffer (50 mmol / L Tris-HCl pH 7.4, 150 mmol / L potassium chloride, 10 mmol / L magnesium chloride) in the presence of 1 mmol / L NADPH at 37°C for 0 or 30 minutes (oxidative reaction). After the reaction, 50 μL of the reaction mixture was added to 100 μL of a 1 / 1 (v / v) methanol / acetonitrile solution, mixed, and centrifuged at 3000 rpm for 15 minutes. The test compound in the supernatant was quantified by LC / MS-MS, and the amount of test compound remaining after the reaction was calculated based on the amount of compound at 0 minutes.

[0141] [Metabolic stability test] Using various commercially available frozen hepatocytes, the target compound is reacted for a certain period of time, and the residual rate is calculated by comparing the reacted sample with the unreacted sample, thereby evaluating the degree of metabolism in the liver.

[0142] Various frozen liver cells 1.0x10 6 The reaction mixture was incubated at 37°C for 0, 1, or 2 hours in William's E medium containing 100 cells / mL. After the reaction, 120 μL of a 1 / 1 (v / v) methanol / acetonitrile solution was added to 30 μL of the reaction mixture, mixed, and centrifuged at 3000 rpm for 15 minutes. The test compound in the supernatant was quantified by LC / MS-MS, and the amount of test compound remaining after the reaction was calculated based on the amount of compound at 0 minutes of reaction.

[0143] [Protein binding test] Using various types of serum, the serum protein unbinding rate of the compound of the present invention is measured.

[0144] The reaction conditions were as follows: evaluation method, equilibrium dialysis; reaction time, 24 hours; reaction temperature, 37°C; concentration of the compound of the invention, 2 μg / mL.

[0145] The test solution is added to each type of serum and stirred to prepare serum samples with the above compound concentrations. The serum sample is added to one side of an equilibrium dialysis cell, and phosphate-buffered saline (PBS) is added to the other, and equilibrium dialysis is performed at 37°C for 24 hours. The amount of compound in the sample collected from each cell is measured by LC / MS-MS.

[0146] [Formulation example] Injection The compound of the present invention is dissolved in water or a buffer solution to prepare an injection. Depending on the osmotic pressure, physiological saline, phosphate buffer, monosaccharides, disaccharides, and other carbohydrates may also be added.

Claims

1. A compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof: 【Chemical 33】 Here, in formula (I), R １ , R ２ , R ３ , and R ４ are independently H, halogen, hydroxy, cyano, C1-6 alkyl, C1-6 alkoxy, benzyloxy, C1-6 alkoxyC1-6 alkyl, nitro, C1-6 haloalkyl, aminocarbonyl, C1-C6 alkylaminocarbonyl (CONR ８ R ９ (R ８ , R ９ independently represent H or C1-6 alkyl), C1-C6 alkoxycarbonyl, C1-C6 alkylcarbonyl, COOR １０ (R １０ is H or C1-6 alkyl, amino, alkylamino (NR １１ R １２ (R １１ R １２ each independently represents H or C1-6 alkyl), haloalkylsulfanyl, haloalkylsulfinyl, haloalkylsulfonyl, C1-6 alkylthio, C1-6 alkylsulfinyl, C1-6 alkylsulfonyl, aminosulfonyl, sulfo, or sulfamoyl; R ５ represents H, hydroxy, C1-6 alkyl, or halogen; R ６ represents H or C1-6 alkyl; R ７ is a boronic acid (-B(OH) ２ ), representing either a boronic acid ester or a boronic acid amide (However, R １ , R ２ , R ３ , and R ４ When all are H, or R １ , R ２ , R ３ , and R ４ When any one of R is hydroxy, C1-3 alkoxy or methylcarbonyl, R ６ represents C1-6 alkyl)

2. The R ６ 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein represents H or methyl.

3. The R ７ is boronic acid (B(OH) ２ 3. The compound of claim 1 or 2, wherein R represents a boronic acid or a pinacol ester thereof, or a pharmaceutically acceptable salt thereof.

4. The R ５ The compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein

5. The R １ , R ２ , R ３ , and R ４ Any one or more of the following are independently selected from Cl, F, C1-4 alkyl, C1-4 alkoxy, CH ２ X, CHX ２ , or CX ３ (X represents F) or a pharmaceutically acceptable salt thereof.

6. A drug for BNCT, comprising the compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof.

7. A radioisotope-containing diagnostic agent comprising the compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof.

8. A drug for injection and infusion for BNCT, comprising a compound represented by the following formula (I') or a pharmaceutically acceptable salt thereof, and having a total concentration of fructose and sugar alcohol of 0.1% by mass or less: 【Chemical Formula 34】 Here, in formula (I′), R １ , R ２ , R ３ , and R ４ are independently H, halogen, hydroxy, cyano, C1-6 alkyl, C1-6 alkoxy, benzyloxy, C1-6 alkoxyC1-6 alkyl, nitro, C1-6 haloalkyl, aminocarbonyl, C1-C6 alkylaminocarbonyl (CONR ８ R ９ (R ８ , R ９ independently represent H or C1-6 alkyl), C1-C6 alkoxycarbonyl, C1-C6 alkylcarbonyl, COOR １０ (R １０ is H or C1-6 alkyl, amino, alkylamino (NR １１ R １２ (R １１ R １２ each independently represents H or C1-6 alkyl), haloalkylsulfanyl, haloalkylsulfinyl, haloalkylsulfonyl, C1-6 alkylthio, C1-6 alkylsulfinyl, C1-6 alkylsulfonyl, aminosulfonyl, sulfo, or sulfamoyl; R ５ represents H, hydroxy, C1-6 alkyl, or halogen; R ６ represents H or C1-6 alkyl; R ７ is a boronic acid (-B(OH) ２ ), represents either a boronic acid ester or a boronic acid amide.

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