JP2012153647A - Carborane-modified kojic acid/cyclodextrin inclusion complex and production method of the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a carborane-modified kojic acid/cyclodextrin inclusion complex having excellent solubility with water.SOLUTION: A complex of a cyclodextrin derivative and a carborane-modified kojic acid is prepared. The preparation process includes a step of vibrating a mixture of the cyclodextrin derivative and the carborane-modified kojic acid.

Description

  The present invention relates to a carborane-modified kojic acid / cyclodextrin inclusion complex and a method for producing the same. More specifically, the present invention relates to a complex of cyclodextrin derivative and 7-o-carboranyl kojic acid or kojic acid o-carboranyl methyl ether, particularly for use in boron neutron capture therapy (BNCT), and a method for producing the same. About.

In recent years, boron neutron capture therapy (BNCT) has attracted attention as a cancer treatment method using radioactive isotopes. In boron neutron capture therapy, a boron compound containing a boron 10 isotope ( ¹⁰ B) is taken into a cancer cell, irradiated with a low-energy neutron beam (for example, thermal neutron), and locally by a nuclear reaction occurring in the cell. It is a treatment method that destroys cancer cells. In this method of treatment, it is important to increase the therapeutic effect by selectively accumulating boron compounds containing 10B in cancer tissue cells. Therefore, a boron compound that is selectively and reliably taken up by cancer cells is developed. It is necessary to do.

  Conventionally, various boron-containing compounds in which a boron atom or a boron atom group is introduced into a basic skeleton have been synthesized as drugs used for BNCT. So far, drugs used in actual clinical practice include p-boronophenylalanine (BPA) and mercaptoundecahydrododecaborate (BSH).

  On the other hand, carborane and the like are attracting attention as other compounds. However, since carborane is hardly soluble in water and does not have cell selectivity, it is difficult to introduce carborane into cells or to accumulate around cancer cells. For this reason, various studies have been made so far, such as improvement of cell selectivity, water solubility, and modification of substituents (Non-patent Documents 1-8 and 1).

  In particular, carborane derivatives with a kojic acid structure, which is known for its ability to accumulate cells in melanocytes and is expected to act as a cell-introducing ligand, have been synthesized. It has not reached (Non-Patent Documents 9 to 10).

JP 2008-222585 A

S. B. Kaul, R.A. Kaser, J. Am. Chem. Soc., 118, 1223 (1996). P. D. Godfrey, W. J. Crigsby, P. J. Nichols, C. L. Raston, J. Am. Chem. Soc., 119, 9283 (1997). A. Harada, S. Takahashi, J. Chem. Commun., 12, 1352 (1988). M-J. Hardie, C-L. Raston., J. Chem. Commun., 12, 1153 (1999). M-J. Hardie, C-L. Raston., Eur. J. Inorg. Chem., 12, 195 (1999). L. Craciun, R. Custelcean, Inorg. Chem, 38, 4916 (1999). L. Deng, H-S. Chan, Z. Xie, J. Am. Chem. Soc, 128, 5219. (2006). A. H. Soloway, W. T. Beverly, A, Barnum, F-G. Rong, R-F. Barth, I-W. Codogni, J. G. Wilson, Chem. Rev., 98, 1515 (1998). S. Parves, M.K.Kang, H.S.Chung, C.W.Cho, M.C.Hong, M.K.Shin, H.Bae, PHYTOTHER. Res., 20, 921 (2006) H. Nakao, M. Kirihata, M. Kobayashi, Y. Kitaoka, Adv. Neutron Capture Ther., 1132, B126 (1997)

  A carborane derivative containing kojic acid has low water solubility, and thus it is difficult to evaluate biological activity, and its usefulness is poor.

  Accordingly, an object of the present invention is to provide a carborane-modified kojic acid / cyclodextrin inclusion complex which is excellent in stability and rich in water solubility that can be put to practical use in BNCT.

  Another object of the present invention is to provide a method for producing such a carborane-modified kojic acid / cyclodextrin inclusion complex.

  As a result of intensive studies to solve the above problems, the present inventors have found that a complex of a cyclodextrin derivative and a carborane-kojic acid compound ensures high water solubility, stability, and safety. The present invention has been found to be suitable, and the present invention has been completed.

That is, the present invention provides a cyclodextrin derivative,

(Here, R ¹ represents hydrogen, boron, methyl group or ethyl group, and R ² represents hydrogen, methylcarbonyl or arylcarbonyl group.)
Or

(Wherein R represents hydrogen, arylcarbonyl, or methylcarbonyl).
Here, the black circle in the chemical formula represents C, and the white circle represents BH (the same applies hereinafter).

The present invention also relates to complexes of cyclodextrin derivatives with 7-o-carboranyl kojic acid or kojic acid o-carboranyl methyl ether.

  The cyclodextrin derivative may be one selected from the group consisting of α-cyclodextrin, β-cyclodextrin, methyl-β-cyclodextrin, and hydroxypropyl-β-cyclodextrin.

The present invention also relates to a composition for boron neutron capture therapy comprising the complex.

The present invention also provides a cyclodextrin derivative,

(Here, R ¹ represents hydrogen, boron, methyl group or ethyl group, and R ² represents hydrogen, methylcarbonyl or arylcarbonyl group.)
Or

(Wherein R represents hydrogen, arylcarbonyl, or methylcarbonyl), and relates to a method that includes a step of vibrating or stirring a mixture.

The present invention also provides a method for producing a complex of a cyclodextrin derivative and 7-o-carboranyl kojic acid or kojic acid o-carboranyl methyl ether,
The present invention relates to a production method comprising a step of shaking or stirring a mixture of a cyclodextrin derivative and 7-o-carboranyl kojic acid or kojic acid o-carboranyl methyl ether.

The cyclodextrin derivative may be one selected from the group consisting of α-cyclodextrin, β-cyclodextrin, methyl-β-cyclodextrin, and hydroxypropyl-β-cyclodextrin.

  The complex of the cyclodextrin derivative of the present invention and a carborane-modified kojic acid is excellent in water solubility and stability, has properties acceptable for administration to humans and animals, and is particularly useful for BNCT. Also, the toxicity exhibited by carborane modified kojic acid alone is dramatically reduced.

It is a figure which shows the 1H-NMR spectrum of a solubilizer cyclodextrin and a CKA complex. It is a figure which shows the cell activity with respect to the cultured cell of the complex of o-carborane and methyl-beta-cyclodextrin. It is a figure which shows the cell activity with respect to the cultured cell of the complex of CKA and methyl-beta-cyclodextrin. It is a figure which shows the cell activity with respect to the cultured cell of the complex of CKA and a hydroxypropyl-beta-cyclodextrin. It is a figure which shows the cell activity with respect to the cultured cell of the complex of KACME hydroxypropyl-beta-cyclodextrin. In BNCT, it is a figure which shows the result of BNCT of the cancer-bearing mouse | mouth using the CKA / hydropropyl-beta-cyclodextrin inclusion complex.

  The cyclodextrin derivative referred to in the present invention refers to an oligosaccharide having a structure in which glucose is linked in a cyclic manner, and includes α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, and derivatives thereof. Those methylated or hydroxypropylated derivatives can be used particularly preferably. In particular, it is preferably one selected from the group consisting of α-cyclodextrin, β-cyclodextrin, methyl-β-cyclodextrin, and hydroxypropyl-β-cyclodextrin.

The carborane-modified kojic acid of the present invention is

(Here, R ¹ represents hydrogen, boron, methyl group or ethyl group, and R ² represents hydrogen, methylcarbonyl or arylcarbonyl group.)
Or

(Wherein R represents hydrogen, arylcarbonyl, or methylcarbonyl). Aryl here is preferably phenol, alkyl group monosubstituted, disubstituted, or trisubstituted phenol, or halogen-substituted phenol, but is not particularly limited.

Preferably, it refers to 7-o-carboranyl kojic acid or kojic acid o-carboranyl methyl ether.

In the present invention, a carborane-modified kojic acid / cyclodextrin inclusion complex can be prepared by mixing such a carborane-modified kojic acid and a cyclodextrin derivative in an aqueous solution. Here, the preparation preferably includes a step of shaking or stirring the mixed solution, in particular, a step of preparing a suspension by a high-speed vibration grinding method or a vortex method, both a cyclodextrin derivative and a compound in a polar organic solvent such as DMSO. It is preferable to include a solvent replacement method in which is dissolved and replaced with an aqueous solution. Furthermore, when the liquid thus obtained is centrifuged, for example, and the supernatant is recovered, the solubilized complex can be recovered.

In addition, it is also possible to prepare the complex of the present invention by mixing compounds in a solid state as they are, vibrating or mixing them.

Furthermore, it is possible to employ any of various methods for producing a complex. Examples of the method for producing such a complex include spray drying, freeze drying, saturated aqueous solution method, kneading method, and mixing and pulverizing method. Also included.

For example, the saturated aqueous solution method is a method in which carborane-modified kojic acid is introduced into a saturated aqueous solution of a cyclodextrin derivative and mixed with stirring. In the kneading method, a cyclodextrin derivative, water and a carborane-modified kojic acid are kneaded to obtain an inclusion complex. In the spray drying method, a cyclodextrin derivative or an aqueous polysaccharide solution containing a cyclodextrin derivative and a carborane-modified kojic acid are spray-dried to obtain a powder containing an inclusion complex.

In any case, industrially, an apparatus having a combination of a blade, a rotating blade, and a screw, a ball mill, a V-type mixer, a screw mixer, and the like can be used.

As a solubilizer for carborane-modified kojic acid, in addition to cyclodextrin derivatives, polysaccharides (β-1,3-glucan, dextran), oligosaccharides (cycloamylose) and the like can be used.

Here, the aqueous solution is preferably water, but may include an aqueous solution containing a solvent, ethanol, DMSO, glycerol, ethylene glycol, polyethylene glycol, and the like.

The carborane modified kojic acid such as 7-o-carboranyl kojic acid or kojic acid o-carboranyl methyl ether and the cyclodextrin derivative are in a weight ratio of 1: 2 to 1:10, preferably 1: 3 to 3. The carborane-modified kojic acid / cyclodextrin inclusion complex of the present invention can be prepared by containing it in an aqueous solution at a ratio of 1: 5.

A complex of a carborane-modified kojic acid such as 7-o-carboranyl kojic acid or kojic acid o-carboranyl methyl ether and a cyclodextrin derivative, depending on the amount ratio, for example, at least 20 to 20 in water. 50 ppm can be present. When the amount of cyclodextrin derivative is 3 times or more, preferably 5 times or more of 7-o-carboranyl kojic acid or kojic acid o-carboranyl methyl ether, 100 ppm or more, 3000 ppm depending on conditions ˜4000 ppm can be present.

In the present invention, a composition for boron neutron capture therapy containing the complex thus prepared can be prepared.

The boron neutron capture therapy composition of the present invention may be in any form, but is preferably a liquid. When it is liquid, it may be aqueous, alcoholic, or any other liquid, but is preferably an aqueous liquid.

The pH when the composition of the present invention is liquid is preferably a neutral pH in consideration of administration to a living body. More specifically, it is in the range of 4 to 8, preferably 6.5 to 7.5, particularly around 7.4. However, a lower pH may be used when used as an oral preparation. For the adjustment, if necessary, an appropriate pH adjusting agent (hydrochloric acid, sodium hydrogen carbonate, etc.) used in this technical field, a buffering agent or the like may be used.

The osmotic pressure ratio of the composition of the present invention is not particularly limited, but it is preferably in the range of 1 to 2 with respect to physiological saline. More preferably, it is in the range of 1.1 to 1.4. When it is within this range, in the case of an injection, pain can be reduced and administration time can be shortened.

In the composition of the present invention, various metal ions that may be contained in a living body may be appropriately contained in order to achieve stability inside and outside the living body. Preferably, sodium ions are contained, and the concentration thereof is not particularly limited, but is particularly preferably from 130 mEq / L to 160 mEq / L. This numerical range close to the Na ion concentration range of the body fluid is preferable so that the electrolyte balance between the intracellular fluid and the extracellular fluid is not greatly lost.

If necessary, a buffering agent such as a phosphate buffer, a Tris-HCl buffer, an acetate buffer, a carbonate buffer, or a citrate buffer may be added to the composition of the present invention. These buffers may be useful for stabilizing the formulation or reducing irritation.

Furthermore, the composition of the present invention can contain other components that are usually used in the technical field, if necessary, as long as the object of the present invention is not adversely affected. As such components, additives usually used in liquid, especially aqueous compositions, for example, preservatives such as benzalkonium chloride, potassium sorbate, chlorohexidine hydrochloride, stabilizers such as sodium edetate, hydroxyethyl cellulose , Thickeners such as hydroxypropylmethylcellulose, isotonic agents such as sodium chloride, potassium chloride, glycerin, sucrose, glucose, surfactants such as polysorbate 80, polyoxyethylene hydrogenated castor oil, sodium chloride, potassium chloride, Examples thereof include isotonic agents such as glycerin and pH adjusting agents such as hydrochloric acid and sodium hydroxide.

The composition of the present invention can take the form of nasal drops, oral preparations, vaginal preparations, suppositories, injections and the like as pharmaceuticals used for boron neutron therapy.

That is, when the composition of the present invention is used as a pharmaceutical, it is any form of oral administration by liquid or the like, or parenteral administration by injection such as intraarterial injection, intravenous injection or intramuscular injection, suppository, or transdermal preparation It may be. The parenteral administration includes intravenous, intramuscular, subcutaneous, intra-tissue, intranasal, intradermal, instillation, intracerebral, rectal, intravaginal, intraperitoneal administration, and the like.

The preparation can be in any form such as powder, granule, fine granule, dry syrup, tablet, capsule, injection, dry solid for injection, liquid and the like. Depending on the dosage form, appropriate excipients; disintegrating agents; binders; lubricants; diluents; phosphoric acid, citric acid, succinic acid, acetic acid, and Buffering agents such as organic acids or their salts; isotonic agents; preservatives; wetting agents; emulsifiers; dispersants; stabilizers; solubilizers; antioxidants such as ascorbic acid; Polypeptides (eg, less than about 10 residues) (eg, polyarginine or tripeptides); proteins (eg, serum albumin, gelatin, or immunoglobulin); hydrophilic polymers (eg, polyvinylpyrrolidone); amino acids (eg, glycine, glutamic acid) , Aspartic acid, or arginine); monosaccharides, disaccharides and other carbohydrates (cellulose or derivatives thereof, glucose, mannose, or dextrin) Pharmaceutical additives such as chelating agents (eg, EDTA); sugar alcohols (eg, mannitol or sorbitol); counterions (eg, sodium); and / or nonionic surfactants (eg, polysorbates, poloxamers) It can be prepared by appropriately mixing or diluting / dissolving with the product. Such substances that enhance isotonicity and chemical stability are non-toxic to recipients at the dosages and concentrations used.

When the dosage form is oral, the composition can be solid or liquid, but it is particularly preferred that it be a liquid, in which case the pharmaceutically acceptable emulsions, solutions, suspensions of the above materials are particularly preferred. Agents, syrups, elixirs and the like, and generally used inert solvents such as purified water and ethanol. In addition to the inert solvent, this composition may contain adjuvants such as solubilizers, wetting agents, and suspending agents, sweeteners, corrigents, fragrances, and preservatives.

When the injection for parenteral administration is liquid, a certain amount of active ingredient is dispersed in a dispersant (for example, polysorbate 80, polyoxyethylene hydrogenated castor oil 60, polyethylene glycol, carboxymethylcellulose, sodium alginate, etc.), preservative (For example, methylparaben, propylparaben, benzyl alcohol, chlorobutanol, phenol, etc.), isotonic agents (for example, sodium chloride, glycerin, D-mannitol, glucose, etc.) and the like, and aqueous solvents (for example, distilled water for injection, physiological For example, olive oil, sesame oil, cottonseed oil, vegetable oils such as corn oil, propylene glycol, etc.) and the like. At this time, additives such as a solubilizing agent (for example, sodium salicylate, sodium acetate, etc.), a stabilizer (for example, human serum albumin), a soothing agent (for example, benzyl alcohol, etc.) may be used as desired. Furthermore, you may contain an antioxidant, a coloring agent, etc. and another additive as needed.

A “pharmaceutically acceptable carrier” can also be used. Examples of such substances include solvents, solubilizers, suspending agents, isotonic agents, buffers, soothing agents and the like in liquid preparations. If necessary, formulation additives such as preservatives, antioxidants, colorants, sweeteners, adsorbents, gelling agents and the like can be used according to a conventional method.

Preferable examples of the “antioxidant” include sulfite and ascorbic acid. Preferable examples of the “isotonic agent” include glucose, sodium chloride, glycerin, D-mannitol and the like.

Preferable examples of the “solubilizing agent” include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate and the like. .

Preferable examples of the “solvent” include water for injection, alcohol, propylene glycol, macrogol and the like.

Preferable examples of the “suspending agent” include hydrophilic polymers such as polyvinyl alcohol, polyvinyl pyrrolidone, sodium carboxymethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose and the like.

Examples of the “surfactant” include sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glyceryl monostearate and the like.
Preferable examples of the “soothing agent” include benzyl alcohol and the like.

Preferable examples of the “preservative” include p-hydroxybenzoates, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like.

Techniques for prescribing and administration are described, for example, in the latest edition of the Japanese Pharmacopoeia and the latest supplement, “REMINGTON'S PHARMACEUTICAL SCIENCES” (Maack Publishing Co., Easton, PA).

EXAMPLES The present invention will be described in more detail with reference to the following examples, but these do not limit the scope of the present invention.

  In the methods shown in the following examples, first, 7-o-carboranyl kojic acid and kojic acid o-carboranyl methyl ether obtained in Production Example 1 were synthesized, and further, using these carborane-modified kojic acids. A carborane modified kojic acid / cyclodextrin inclusion complex was prepared. Then, solubilization rate and toxicity evaluation were performed for these carborane-modified kojic acid / cyclodextrin inclusion complexes.

(Production Example 1) Synthesis of 7-o-carboranyl kojic acid (CKA) The 7-o-carbonyl kojic acid (CKA) of the present invention refers to a compound having the following structure.

(1) Synthesis of o-carboranyl tributyltin (1)


o-Carborane (100 mg, 0.70 mmol) was dissolved in anhydrous tetrahydrofuran (8 ml) and cooled to −78 ° under an argon stream. To this, a tetrahydrofuran solution (0.46 ml, 0.78 mmol) of n-butyllithium was added dropwise over 30 minutes, and stirring was continued for 90 minutes at the same temperature. To this reaction solution, n-tributyltin chloride (0.20 ml, 0.72 mmol) was added dropwise over 10 minutes and stirred at the same temperature for 5 minutes. The cooling bath was removed and the reaction temperature was raised to room temperature and concentrated under reduced pressure. Water and diethyl ether were added to the obtained crude product for extraction. The organic layer was washed successively with water and saturated brine, and then dried over anhydrous sodium sulfate, and diethyl ether was distilled off under reduced pressure. The crude product was purified using a silica gel column to give pure o-carboranyltributyltin (270 mg, 90%) as an oil.

Analysis value
¹ H-NMR (CDCl ₃ ) δ: 0.91 (9H, t, J = 7.2 Hz, CH ₃ ), 0.99-3.10 (37H, m), 3.23 (1H, s)
¹³ C-NMR (CD ₃ OD) δ: 11.8, 13.5, 27.2, 28.3
IR (cm ^-1 ): 2961, 2921, 2873, 2855, 2593, 1463, 1417, 1378, 1072, 1021, 721, 673

(2) Synthesis of kojic acid benzyl ether (2)
(5-Benzyloxy-2-hydroxymethyl-4H-pyran-4-one)

Under a nitrogen stream, kojic acid (14.2 g, 0.1 mol) was added to a sodium methylate solution prepared from anhydrous methanol (200 ml) and metallic sodium (2.3 g, 0.1 atm), and the mixture was stirred at room temperature for 2 hours. To this solution, benzyl chloride (13 ml, 0.11 mmol) was added dropwise and heated to reflux for 3 hours. Next, 500 ml of water was added to the reaction solution under ice cooling, and the precipitated crystals were collected by suction filtration. The crude crystals were recrystallized from ethanol to obtain 2 (13 g, 74%) as colorless needle crystals.

As another method, the following method was also possible.

To a methanolic solution (140 ml) of kojic acid (20.0 g, 0.14 mol), an aqueous sodium hydroxide solution (14 ml, 6.2 g, 0.155 mo) was added, and benzyl chloride (19.7 g, 0.155 mol) was added dropwise under reflux with heating. The reflux continued all day and night. The reaction mixture was concentrated under reduced pressure, water (200 ml) was added to the residue, and the solid was collected by suction filtration. The crude crystals were recrystallized from methanol to obtain 2 (23.0 g, 71%) as colorless needle crystals.

Analysis value of 2
Mp: 131-133 ℃
¹ H-NMR (CDCl ₃ ) δ: 3.73 (1H, t, J = 6.5 Hz, -OH), 4.43 (2H, d, J = 6.5 Hz, -CH ₂ O-), 5.03 (2H, s, PhCH _2- ), 6.52 (1H, s, 3-H), 7.29-7.38 (5H, m, C ₆ H ₅ ), 7.51 (1H, s, 6-H)
¹³ C-NMR (CD ₃ OD) δ: 60.9, 72.4, 111.9, 129.1, 129.3, 129.5, 137.1, 142.9, 148.2, 170.5, 176.8
IR (cm ^-1 ): 3323, 3112, 2906, 2838, 1646, 1605, 1502, 1457, 1445, 139, 1329, 1261, 1236, 1207, 1151, 1083, 1021, 949, 867, 839, 779, 744 , 697, 587, 535

3) Synthesis of 5-benzyloxy-2-formyl-4H-pyran-4-one (3)
(5-Benzyloxy-2-formyl-4H-pyran-4-one)

Manganese dioxide (100 g) was added to a solution of 2 (13 g, 56 mmol) in benzene (90 ml), and the mixture was heated to reflux for 4 hours. The reaction solution was suction filtered using Celite, and the filtrate was concentrated under reduced pressure to obtain a crude product, which was recrystallized from ethyl acetate-hexane to give aldehyde 3 (11 g, 83%) as a colorless prism. Obtained as a crystal.

Alternatively, the following method was also possible.

To a solution of 2 (10.0 g, 43 mmol) in chloroform (200 ml) were added triethylamine (36.8 ml, 265 mmol) and dimethyl sulfoxide (54 ml) and cooled to 5 ° C. To this was added sulfur trioxide pyridine complex (34.2 g, 215 mmol), and stirring was continued overnight at room temperature. The reaction solution was washed successively with water and saturated brine, dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column to give pure 3 (8.1 g, 81%) as colorless crystals.

Analysis value of 3
Mp: 120-121 ° C ¹ H-NMR (CDCl ₃ ) δ: 5.13 (2H, s, PhCH ₂ ), 6.99 (1H, s, 3-H), 7.34-7.40 (5H, m, C ₆ H ₅ ) , 7.66 (1H, s, 6-H), 9.64 (1H, s, CHO)
¹³ C-NMR (CD ₃ OD) δ: 72.1, 120.1, 127.9, 128.8, 129.0, 135.1, 141.4, 141.5, 141.6, 149.2, 155.7, 174.2, 184.0
IR (cm ^-1 ): 3100, 2852, 1718, 1656, 1620, 1589, 1500, 1463, 1455, 1392, 1299, 1211, 1191, 1143, 1018, 939, 870, 744

(4) Synthesis of 7-o -carboranyl kojic acid benzyl ether (4) (5-Benzyloxy-5- (1'-o-carboranyl-1'-hydroxy) methyl-4H-pyran-4-one)


1 (0.7 g, 1.6 mmol), 3 (0.29 g, 1.2 mmol) and tetrahydrofuran (15 ml) were placed in a synthetic flask replaced with an argon stream and stirred. To this solution, tris (dibenzylideneacetone) dipalladium chloroform (0.28 g, 0.27 mmol) and 1,2-bisdiphenylphosphinoethane DPPE (0.21 g, 0.54 mmol) were added and refluxed for 3 hours. The reaction solution was concentrated under reduced pressure, and the resulting crude product was purified by a silica gel column to give pure 4 (0.45 mg, 94%) as colorless prism crystals.

Analysis value of 4
Mp: 213-216
¹ H-NMR (CDCl ₃ ) δ: 1.00-3.30 (11H, m, carborane), 4.79 (1H, br s, OH), 5.12 (1H, s, 7-H), 5.14 (2H, s, PhCH ₂ ), 6.69 (1H, s, 3-H), 7.40-7.56 (5H, m, C ₆ H ₅ ), 8.18 (1H, s, 6-H)
¹³ C-NMR (CD ₃ OD): δ: 60.9, 71.7, 72.6, 77.9, 113.7, 129.3, 129.6, 137.0, 142.7, 148.9, 167.1, 176.4
IR (cm ^-1 ): 3298, 3119, 3101, 3050, 2940, 2871, 2603, 2581, 1642, 1597, 1455, 1257, 1213, 1164, 1109, 988, 961, 833, 749, 700

(5) Synthesis of 7-o-carboranyl kojic acid (CKA)
(7-o-Caboranylkojic acid)
{2- (1'-o-Carboranyl-1'-hydroxy) methyl-5-hydroxy-4H-pyran-4-one}


A methanol solution of 4 (100 mg, 0.26 mmol) to which 10% -palladium carbon (30 mg) was added was attached to a medium pressure reducing device, and stirred at room temperature and 4 kg / cm ² under hydrogen pressure for 4 hours. The palladium carbon catalyst was removed by suction filtration, and the filtrate was concentrated under reduced pressure to obtain a crude product. This was purified by a silica gel column to give CKA (270 mg, 90%) as colorless prism crystals.

Analysis value of CKA
Mp: 98-100 ℃
¹ H-NMR (CDCl ₃ ): δ: 1.00-3.30 (11H, m, carborane), 4.78 (1H, m, CH ₂ O),
5.12 (1H, s, CH), 6.67 (1H, s, 3-H), 8.10 (1H, s, 6-H)
¹³ C-NMR (CD ₃ OD) δ: 60.9, 71.8, 78.0, 112.4, 140.6, 148.2, 166.9, 176.3
IR (cm ^-1 ): 3285, 3085, 2975, 2925, 2599, 1724, 1614, 1572, 1466, 1394,
1256, 1213, 1158, 1077, 875, 834, 718
FABMS: m / z 285 (M ⁺ +1)

(Production Example 2)
II. Synthesis of kojic acid o-carboranyl methyl ether (KACME)
{Synthesis of 5- (o-Carboranyl) methoxy-2-hydroxymethyl-4H-pyran-4-one}
Kojic acid o-carboranyl methyl ether is a compound having the following structure.

(1) Synthesis of kojic acid propynyl ether (5)
{2-Hydroxymethyl-5-propynyloxy-4H-pyran-4-one}

To a sodium methylate solution prepared from anhydrous methanol (100 ml) and metallic sodium (0.80 g, 35 atm), kojic acid (5.00 g, 35 mmol) was added under a nitrogen stream and stirred for 90 minutes. 3-Bromopropyne (3.44 ml, 38.7 mmol) was slowly added dropwise to the reaction solution in which a white precipitate was formed. After completion of the addition, the mixture was heated and stirred at 60 ° C. for 8 hours. The reaction solution was concentrated under reduced pressure to obtain a yellow oily residue. Water (15 ml) was added thereto, and the mixture was extracted with ethyl acetate (20 ml × 10). The organic layer was washed with saturated brine, dried over sodium sulfate and concentrated, and the obtained oil was purified by a silica gel column to give pure 5 (5.90 g, 93%) as crystals.

Analysis value of 5
Mp: 90-92 ° C
¹ H-NMR (CDCl ₃ ) □: 2.58 (1H, t, J = 2.4 Hz, CH), 4.50 (2H, s, J = 5.8 Hz, CH ₂ O),
4.73 (2H, d, J = 2.4 Hz, CH ₂ C), 4.85 (2H, t, J = 6.4 Hz, OH),
6.55 (1H, s, 3-H), 7.82 (1H, s, 6-H)
¹³ C-NMR (CD ₃ OD): 57.4, 60.1, 76.2, 76.6, 111.7, 142.6, 145.2, 168.1, 174.9 IR (cm ^-1 ): 3279, 3256, 2134, 1646, 1607, 1591, 1230, 1205, 1161, 1034,
1002, 972, 923, 878, 745, 673

(2) Synthesis of 2-Benzoxymethyl-5-propynyloxy-4H-pyran-4-one (6)

  5 (2.00 g, 11 mmol), a mixed solution of dimethylaminopyridine (0.10 g) and pyridine (50 ml) was cooled to 0 ° C., and benzoyl chloride (1.35 ml, 11 mmol) was slowly added dropwise thereto. After completion of the dropwise addition, the reaction solution was returned to room temperature (20 ° C.) and stirred for 1 hour. Concentrated under reduced pressure to remove excess pyridine, and the residual oil was poured into dilute hydrochloric acid. This was extracted with ethyl acetate, the organic layer was washed with saturated brine, dried over sodium sulfate, and the solvent was distilled off. The obtained residue was purified with a silica gel column (ethyl acetate: hexane = 2: 8-3: 7) to obtain 6 (2.84 g, 90%) as crystals.

Analysis value of 6
Mp: 74 ℃
¹ H-NMR (CDCl ₃ ): 2.58 (1H, t, J = 2.4 Hz, CH), 4.78 (2H, d, J = 2.4 Hz, CH ₂ C),
5.17 (2H, s, CH ₂ OBz), 6.55 (1H, s, 3-H), 7.48 (2H, t, J = 7.6
Hz, mC ₆ H ₅ ), 7.62 (1H, t, J = 7.3 Hz, pC ₆ H ₅ ), 7.85 (1H, s, 6-H),
8.08 (2H, d, J = 7.6 Hz, oC ₆ H ₅ )
¹³ C-NMR (CD ₃ OD): 57.8, 61.2, 77.1, 77.2, 114.3, 128.5, 128.6, 129.8, 133.7,
143.2, 145.9, 161.9, 165.4, 174.2
IR (cm ^-1 ): 3243, 3081, 2135, 1725, 1645, 1615, 1595, 1453, 1439, 1266,
1249, 1215, 1166, 1107, 1096, 1071, 995, 970, 921, 884, 732,
717, 702, 690

(3) Synthesis of 2-Benzoxymethyl-5-o-carboranylmethoxy-4H-pyran-4-one (7)


Under an argon stream, 6 (0.55 g, 1.9 mmol) and decaborane (0.24 g, 1.9 mmol) were dissolved in anhydrous propionitrile (6 ml) and stirred at 20 ° C. for 4 hours and then at 60 ° C. for 12 hours. The reaction mixture was concentrated under reduced pressure to give a crude product, which was purified by silica gel column (ethyl acetate: hexane = 2: 8) to give pure 7 (0.24 g, 32%) as colorless crystals.

Analysis value of 7
Mp: 122 ° C
¹ H-NMR (CDCl ₃ ): 1.103.30 (10H, m, BH), 4.31 (1H, broad, carborane CH),
4.46 (2H, s, CH ₂ ), 5.16 (2H, d, J = 0.6 Hz, CH ₂ OBz), 6.53 (1H,
s, 3-H), 7.45 7.51 (2H, m, mC ₆ H ₅ ), 7.59 7.66 (1H, m,
pC ₆ H ₅ ), 7.80 (1H, s, 6-H), 8.048.08 (2H, m, oC ₆ H ₅ )
¹³ C-NMR (CD ₃ OD): 58.3, 61.2, 71.9, 115.0, 128.5, 128.6, 129.8, 133.8, 145.3,
146.5, 162.5, 165.4, 173.9
IR (cm ^-1 ): 3220, 3071, 2597, 1731, 1663, 1628, 1455, 1270, 1233, 1210,
1177, 1105, 1095, 1028, 709

(4) Synthesis of kojic acid o-carboranyl methyl ether (KACME)
{5- (o-Carboranyl) methoxy-2-hydroxymethyl-4H-pyran-4-one}

  To a solution of 7 (1.00 g, 2.48 mmol) in anhydrous methanol (20 ml) cooled to 0 ° C., 90% sodium methoxide (0.15 g, 2.48 mmol) was added and stirred at the same temperature for 5 minutes, then 20 ° C. And stirring was continued for 50 minutes. The reaction solution was adjusted to pH 7 by adding 0.2N hydrochloric acid, and extracted with ethyl acetate (50 ml × 2). The organic layer was washed with saturated brine, dried over sodium sulfate and concentrated under reduced pressure to obtain a crude product. This was purified by a silica gel column (ethyl acetate: hexane = 1: 1) to obtain pure KACME (0.59 g, 80%).

Analysis value of KACME
Mp: 136 ° C
¹ H-NMR (CDCl ₃ ): 1.10-3.30 (10H, m, BH), 2.67 (1H, t, J = 6.3 Hz, OH),
4.34 (1H, broad s, carborane CH), 4.44 (2H, s, CH ₂ ), 4.50
(2H, d, J = 5.5 Hz, CH ₂ OH), 6.52 (1H, s, 3-H), 7.78 (1H, s,
6-H)
¹³ C-NMR (CD ₃ OD): 58.2, 60.6, 71.7, 71.9, 113.1, 145.0, 146.3, 167.5, 174.5
IR (cm ^-1 ): 3299, 3058, 2608, 2583, 2553, 1659, 1642, 1619, 1593, 1449,
1266, 1210, 1154, 1083, 1030, 862, 725
FABMS: m / z 299 (M ⁺ +1)

[Example 1]
Solubilization of kojic acid-modified carborane compounds by high-speed vibration grinding method α-cyclodextrin (α-CD, manufactured by Wako Pure Chemical Industries), β-cyclodextrin (β-CD, manufactured by Wako Pure Chemical Industries), methyl -β-cyclodextrin (Me-β-CD, manufactured by Wako Pure Chemical Industries, Ltd.), hydroxypropyl-β-cyclodextrin (HP-β-CD, manufactured by Wako Pure Chemical Industries, Ltd.), cycloamylose (CAm, manufactured by Wako Pure Chemical Industries, Ltd.) ), Schizophyllan (SPG, manufactured by Mitsui Sugar Co., Ltd.), dextran (manufactured by Wako Pure Chemical Industries, Ltd.), o-carborane or kojic acid modified o-carborane derivative (CKA, KACME) are mixed in a predetermined amount shown in Table 1, It was enclosed in an agate grinding jar together with agate grinding balls and subjected to high-speed vibration grinding at 25 Hz for 20 minutes in a Lecce mixer mill (MM200). Thereafter, the pulverized product was extracted with the amount of MilliQ water shown in Table 1 and centrifuged (25 ° C., 3,000 rpm, 10 min) to separate the supernatant. Boron concentration was measured with an ICP emission spectrometer (Vista-MPX, manufactured by SII Nanotechnology) to evaluate the solubilization rate.

  As shown in the results of Table 1, even when any solubilizing agent was used, an aqueous solution having a higher boron concentration was successfully produced in all boron compounds than by dissolving the boron compound alone. In particular, when methyl-β-cyclodextrin or hydroxypropyl-β-cyclodextrin having high water solubility of the solubilizer itself is used, o-carborane is about 1160 times, CKA is about 150 times, and KACME is 390 times. It was revealed that solubilizers are effective in increasing water solubility up to double concentration.

[Example 2] Water-solubilization of kojic acid-modified carborane compound by vortex mixing method α-cyclodextrin (α-CD, manufactured by Wako Pure Chemical Industries), β-cyclodextrin (β-CD, Wako Pure Chemical Industries, Ltd.) as water-solubilizing agents ), Methyl-β-cyclodextrin (Me-β-CD, Wako Pure Chemical Industries), hydroxypropyl-β-cyclodextrin (HP-β-CD, Wako Pure Chemical Industries), cycloamylose (CAm, Wako) Table 2 shows o-carborane or kojic acid-modified o-carborane derivatives (CKA, KACME) and MilliQ water using Kosumi Pharmaceutical Co., Ltd., Sizophyllan (SPG, Mitsui Sugar Co., Ltd.), and Dextran (Wako Pure Chemicals Co., Ltd.). The mixture was mixed at the indicated amount and mixed for 60 minutes with a vortex mixer (VORTEX-GENIE 2 manufactured by Scientific Industries). Thereafter, the suspension was centrifuged (25 ° C., 3,000 rpm, 10 min) to separate the supernatant. Boron concentration was measured with an ICP emission spectrometer (Vista-MPX, manufactured by SII Nanotechnology) to evaluate the solubilization rate.

The results are shown in Table 2. Compared with the results of the high-speed vibration pulverization method performed under the same solute-solvent ratio, water-solubilization by the vortex mixing method succeeded in producing an aqueous solution having a concentration of about 1.3 times and in the case of KACME, about 1.2 times higher. It became clear that the vortex method, which is inexpensive to use and easy to operate, is more complex and suitable for preparing aqueous solutions.

[Example 3] Confirmation of CKA / cyclodextrin complex formation by ¹ H-NMR To CKA (5 mg), methyl-β-cyclodextrin (23 mg) or hydroxypropyl-β-cyclodextrin (26 mg) was added. After mixing, heavy water (500 ml) was added, and a complex was prepared by vortex mixing. Then, ¹ H-NMR measurement was performed using an AVANCE 300N nuclear magnetic resonance spectrometer manufactured by Bruker. FIG. 1 shows spectra of Me-β-CD alone (a), Me-β-CD + CKA (b), HP-β-CD alone (c), and HP-β-CD + CKA (d).
In the spectrum in the presence of CKA shown in Fig. 1-b and d, alkenyl protons of kojic acid skeleton are observed as two sharp singlet peaks around 6.7 and 8.2 ppm, and as a very broad signal around 1-3 ppm A proton peak corresponding to 11 hydrogen atoms at the o-carborane site appears, confirming that CKA is water-solubilized in both methyl-β-cyclodextrin and hydroxypropyl-β-cyclodextrin. In addition, the proton signal derived from glucose that constitutes the cyclodextrin ring structure that appears as a complex peak around 3.2-4 ppm has a different signal shape depending on the presence or absence of CKA in both methyl-β-cyclodextrin and hydroxypropyl-β-cyclodextrin. This suggests that CKA interacts with the hydrophobic pores of the structure.

[Example 4] Evaluation of cytotoxicity of kojic acid modified carborane compound / cyclodextrin complex
Colon 26 cells (mouse rectal cancer cells) are seeded in 96-well plates at 1 x 10 ⁵ cells, and used for 24 hours in DME medium (containing 10 w / v% fetal bovine serum) at 37 ° C. The cells were cultured under 5 v / v% CO ₂ . Remove the medium with an aspirator, add 100 mL of medium adjusted to the specified sample concentration, incubate for 22 hours, and use WST reagent (Cell-counting Kit-8, manufactured by Dojindo Laboratories) (see the following document) 10 mL was added and further cultured for 2 hours to perform a color reaction. Using a plate reader (ThermoLab Systems, MultiSkan Acent BIF), the absorbance at 450 nm (reference 650 nm) was measured to calculate the cell viability.

In general, the toxicity of unmodified and simple derivatives is high, and it is known that 1-3 ppm (a fraction of the boron concentration) is toxic (G. Oros, I. Ujvary, RJ). Nachman., Amino Acids, 17 , 357 (1999).). Thus, o-carborane and CKA alone are highly cytotoxic. However, by combining o-carborane and methyl-β-cyclodextrin, the boron concentration, which reduces cell activity against cultured cells by half, was 20 ppm, reducing toxicity (Figure 2). When CKA was used, it was 20 ppm for methyl-β-cyclodextrin and 10 ppm for hydroxypropyl-β-cyclodextrin (FIGS. 3 and 4). Even when KACME was used, it was 20 ppm for hydroxypropyl-β-cyclodextrin (FIG. 5). It is conceivable that a hydrophobic carborane structure is included in the cyclodextrin pores to shield the toxicity. The present invention in which a carborane derivative is water-solubilized using a solubilizing agent has been found to be very advantageous when used as a neutron capture therapy agent.

[Example 5] Tumor growth inhibition evaluation in cancer-bearing mice
(1) Preparation of tumor-bearing mice 6-week-old nude mice (BALB / c Sic-nu / nu mice, male, 18-23 g, obtained from Japan Eslercy) were suspended in Hank's solution under isoflurane inhalation anesthesia. Colon26 cells (1 × 10 ⁵ cell / 100 ml) were subcutaneously administered to the back using a 26G injection needle.

(2) Evaluation of neutron capture effect of CKA / Hp-β-CD complex The tumor-bearing mice obtained as described above were divided into 4 groups as 4 mice per group, and CKA / Hp-β-CD complex Two types of administration methods (two dose groups 12 hours before and 3 hours before neutron irradiation and one dose group 3 hours before) were treated in a total of 4 conditions with or without neutron irradiation, and changes in tumor growth over time Tracked. CKA / Hp-β-CD complex administration was performed by mixing 400 ml of CKA / Hp-β-CD complex aqueous solution with a boron concentration of 375 ppm with 100 ml of 25% glucose solution to make an isotonic solution, 200 ml (boron concentration 300 ppm) was intravenously injected from the tail vein of mice using a 30G needle. Neutron irradiation was performed by the Japan Atomic Energy Agency (JRR? 4) under the conditions of thermal neutron 2 × 10 ¹² fluence / cm ² . Tumor size is measured for 4 weeks after irradiation by measuring the major axis length and minor axis length with electronic calipers, and using the formula: Volume = 1/2 x major axis (mm) x [minor axis (mm)] ² Calculated and evaluated.

  As is clear from the change in tumor volume history (FIG. 6), when the proliferated CKA / Hp-β-CD complex was administered twice and irradiated with neutrons, no change in tumor size was observed even after 4 weeks. A clear growth inhibitory effect was observed. In some cases, the effect of shrinking the tumor to a state where the tumor size cannot be measured is seen, and the kojic acid-modified carborane / cyclodextrin inclusion complex solubilized with a solubilizer is very useful as a BNCT drug. It was found to be a promising complex.

Claims (7)

A cyclodextrin derivative;

(Here, R ¹ represents hydrogen, boron, methyl group or ethyl group, and R ² represents hydrogen, methylcarbonyl or arylcarbonyl group.)
Or

(Wherein R represents hydrogen, arylcarbonyl, or methylcarbonyl) complex. The complex according to claim 1, wherein the complex is a cyclodextrin derivative and 7-o-carboranyl kojic acid or kojic acid o-carboranyl methyl ether.   The complex according to claim 1 or 2, wherein the cyclodextrin derivative is selected from the group consisting of α-cyclodextrin, β-cyclodextrin, methyl-β-cyclodextrin, and hydroxypropyl-β-cyclodextrin. A composition for boron neutron capture therapy comprising the complex according to any one of claims 1 to 3. A cyclodextrin derivative;

(Here, R ¹ represents hydrogen, boron, methyl group or ethyl group, and R ² represents hydrogen, methylcarbonyl or arylcarbonyl group.)
Or

(Wherein R represents hydrogen, a methyl group, an ethyl group, arylcarbonyl, or methylcarbonyl), a method for producing a complex, the method comprising a step of vibrating or stirring the mixture. A method for producing a complex of a cyclodextrin derivative and 7-o-carboranyl kojic acid or kojic acid o-carboranyl methyl ether,
A production method comprising a step of vibrating or stirring a mixture of a cyclodextrin derivative and 7-o-carboranyl kojic acid or kojic acid o-carboranyl methyl ether.   The production method according to claim 5, wherein the cyclodextrin derivative is selected from the group consisting of α-cyclodextrin, β-cyclodextrin, methyl-β-cyclodextrin, and hydroxypropyl-β-cyclodextrin.