Method for producing Lp-boronophenylalanine

Abstract

To provide a method for producing L-p-boronophenylalanine suitable for BNCT therapy.SOLUTION: The present invention provides a method for producing L-p-boronophenylalanine from L-tyrosine through two steps of boronation.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing Lp-boronophenylalanine using L-tyrosine as a raw material, and to a method for producing a pharmaceutical containing Lp-boronophenylalanine obtained by this method as an active ingredient. [Background technology]

[0002] Boron neutron capture therapy (hereinafter referred to as "BNCT") for cancer involves the use of boron neutrons that have been incorporated into tumor tissue in advance. 10 Alpha particles and neutrons produced by the capture reaction of B nuclei with thermal neutrons, which have almost no effect on living organisms. 7 This is a type of radiation therapy that damages tumor cells with Li particles. Lp-boronophenylalanine is known as a drug used in BNCT and is already in clinical use.

[0003] Known methods for producing Lp-boronophenylalanine include a method of producing the DL-isomer of Lp-boronophenylalanine and recovering the L-isomer by optical resolution (Non-Patent Document 1); a method of synthesizing a triflate derivative from a tyrosine derivative raw material in which the amino group is protected as a carbamate ester and the carboxyl group as an alkyl ester, and then reacting the triflate derivative with tetraalkoxydiboron to obtain a compound, and then deprotecting the compound to produce Lp-boronophenylalanine (Patent Document 1); a method of introducing boron into an aromatic amino acid derivative using a borane or diboron compound to produce p-boronophenylalanine (Non-Patent Document 2), and a method for producing its derivatives (Patent Document 2). However, it is not known whether the sensitizing effect of Lp-boronophenylalanine in boron neutron capture therapy differs depending on the production method. On the other hand, the Lp-boronophenylalanine currently used in neutron capture therapy is a high-concentration 10 Since boron (B) is required, expensive compounds using enriched boron have usually been required (Patent Document 3). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] H.R. Synder, Journal of American Chemical Society, 1958, Vol. 80, p. 835 [Non-patent document 2] Yamamoto, Yoshinori et al., Journal of Organic Chemistry, 1998, Vol. 63, pp. 7529-7530 [Patent documents]

[0005] [Patent Document 1] Patent No. 2979139 [Patent Document 2] Patent Publication No. 2000-212185 [Patent Document 3] Patent No. 5150084 Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention provides a simple method for producing Lp-boronophenylalanine, which is suitable for BNCT therapy. [Means for solving the problem]

[0007] The present invention provides [1] A method for producing Lp-boronophenylalanine, comprising the following four steps: First step: reacting L-tyrosine with a boron derivative represented by general formula (1) in a first solvent to produce a boron ester represented by general formula (2); A second step: dissolving the boron ester obtained in the first step in a second solvent and reacting it with trifluoromethanesulfonic anhydride in the presence of a trialkylamine to produce a trifluoromethanesulfonic acid ester represented by the general formula (3); Third step: dissolving the trifluoromethanesulfonic acid ester obtained in the second step in a third solvent and reacting it with a boron compound represented by general formula (4) or a boron compound represented by general formula (5) in the presence of a catalyst to produce an arylboronic acid ester represented by general formula (6); and Fourth step: A step of producing lp-boronophenylalanine by heating the arylboronic acid ester obtained in the third step in a fourth solvent under acidic conditions. [ka] (wherein R1, R2, and R3 are the same or different and represent alkyl, aryl, alkoxy, or aryloxy having 1 to 6 carbon atoms, or R3 represents hydrogen, iodine, methoxy, or trifluoromethanesulfonate, R1 and R2 join together to form a cyclic catechol, pinacol, or bicyclononane, and R4, R5, R6, and R7 are the same or different and represent alkyl, alkoxy, or aryloxy having 1 to 6 carbon atoms), [2] The method for producing Lp-boronophenylalanine according to [1], wherein the boron compound represented by the general formula (4) is bis-pinacolatodiboron. [3] The method for producing Lp-boronophenylalanine according to [1], wherein the boron compound represented by the general formula (5) is pinacolborane. [4] The method for producing Lp-boronophenylalanine according to any one of [1] to [3], wherein the boron derivative represented by the general formula (1) is an alkylborane. [5] The method for producing lp-boronophenylalanine according to [4], wherein the alkylborane is triethylborane or tributylborane. [6] The method for producing Lp-boronophenylalanine according to any one of [1] to [5], wherein the trialkylamine used in the second step is trimethylamine, triethylamine, tributylamine, or diisopropylethylamine. [7] The method for producing Lp-boronophenylalanine according to any one of [1] to [6], wherein the catalyst used in the third step is a palladium catalyst, and the co-catalyst is potassium acetate, ammonium acetate, diisopropylamine, or trimethylamine, triethylamine, or tributylamine, or a combination of two or more thereof. [8] A method for producing lp-boronophenylalanine according to [7], characterized in that the palladium catalyst used is one or more of PdCl2(dppf), PdCl2(dppe), PdCl2(dppp), PdCl2(dppf), CH2Cl2, PdCl2(PPh3)2, PdCl2(o-tolyl)2, and Pd(PPh3)4. [9] A method for producing an enhancer used in tumor radiotherapy, characterized in that the active ingredient is Lp-boronophenylalanine produced by the method according to any one of [1] to [8]; and

[10] The manufacturing method according to [9], wherein the radiation therapy is neutron capture therapy. [Brief explanation of the drawings]

[0008] [Figure 1] Figure 1 shows the time course of changes in tumor volume over 28 days after irradiation in a group of tumor-bearing mice pre-administered with Lp-boronophenylalanine obtained by the present invention and then irradiated with neutrons (L-nBPA-administered group). For comparison, the results of an untreated group and a control group irradiated only with neutrons are also shown. DETAILED DESCRIPTION OF THE INVENTION

[0009] The first step in the present invention, which is a step of reacting L-tyrosine with a boron derivative represented by the general formula (1) in a first solvent to produce a boron ester represented by the general formula (2), refers to the boron esterification step of L-tyrosine shown below.

[0010] [ka] (In the formula, R1, R2, and R3 may be the same or different and represent alkyl, aryl, alkoxy, or aryloxy having 1 to 6 carbon atoms, or R3 represents hydrogen, iodine, methoxy, or trifluoromethanesulfonate, and R1 and R2 together form a cyclic catechol, pinacol, or bicyclononane.)

[0011] Specifically, tyrosine is dissolved in a first solvent, and then, if necessary, under an argon atmosphere, trialkylborane represented by general formula (1) dissolved in the first solvent is added, and the mixture is heated under reflux.

[0012] In the definition of the boron derivative represented by general formula (1), alkyl having 1 to 6 carbon atoms represents, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, etc., aryl represents phenyl, naphthyl, alkoxy represents methoxy, ethoxy, propoxy, butoxy, etc., and aryloxy represents phenyloxy, etc.

[0013] For example, the trialkylborane having alkyl groups of 1 to 6 carbon atoms in R1, R2, and R3 may be any borane having an alkyl group of 1 to 6 carbon atoms, such as trimethylborane, triethylborane, tributylborane, tripropylborane, tripentylborane, and trihexylborane, but it is preferable to use triethylborane or tributylborane, and it is particularly preferable to use triethylborane.

[0014] Furthermore, the expression "R3 represents hydrogen, iodine, methoxy, or trifluoromethanesulfonate, and R1 and R2 join together to form a cyclic catechol, pinacol, or bicyclononane" means, for example, 9-borabicyclo[3.3.1]nonane, in which R3 is hydrogen and R1 and R2 join together to form a bicyclononane; catecholborane, in which R3 is hydrogen and R1 and R2 join together to form a cyclic catechol; and piconalborane, in which R3 is hydrogen and R1 and R2 join together to form a cyclic piconal. oran, 9-iodo-9-borabicyclo[3.3.1]nonane, where R3 is iodine and R1 and R2 together form a cyclic bicycloborane; 9-borabicyclo[3.3.1]nonyltrifluoromethanesulfonate, where R3 is fluoromethanesulfonate and R1 and R2 together form a cyclic bicyclononane; and 9-methoxy-9-borabicyclo[3.3.1]nonane, where R3 is methoxy and R1 and R2 together form a cyclic bicycloborane.

[0015] The first solvent used in this step is an ether solvent, and it is preferable to use tetrahydrofuran or 1,4-dioxane.

[0016] The heating temperature varies depending on the solvent, but it is desirable to reflux at a temperature equal to or higher than the boiling point. The reaction time is 12 to 96 hours, preferably 24 to 72 hours, and particularly preferably 36 to 48 hours.

[0017] The second step in the present invention, which is a step of dissolving the boron ester obtained in the first step in a second solvent and reacting it with trifluoromethanesulfonic anhydride in the presence of a trialkylamine to produce a trifluoromethanesulfonate ester represented by general formula (3), refers to a step of converting a boron ester into trifluoromethanesulfonate as shown below.

[0018] [ka] (In the formula, R1 and R2 may be the same or different and represent alkyl, aryl, alkoxy, or aryloxy having 1 to 6 carbon atoms, or R1 and R2 together form a cyclic catechol, pinacol, or bicyclononane.)

[0019] Specifically, the boron ester represented by the general formula (2) is dissolved in a second solvent, optionally under an argon atmosphere, and a trialkylamine is added. The mixture is cooled and then reacted with trifluoromethanesulfonic anhydride. When the product, trifluoromethanesulfonic acid ester, is confirmed, a basic aqueous solution such as sodium bicarbonate is added to suppress decomposition of the boron ester by the by-product trifluoromethanesulfonate.

[0020] Examples of the second solvent used in this step include acetonitrile, 1,4-dioxane, and cyclopentyl methyl ether, and acetonitrile is particularly preferred. Examples of the trialkylamine used in this step include trimethylamine, triethylamine, tributylamine, and diisopropylethylamine.

[0021] The cooling temperature after the addition of trialkylamine is −20° C. to −60° C., preferably −35° C. to −45° C. The reaction time with trifluoromethanesulfonic anhydride is 15 to 60 minutes, preferably 20 to 40 minutes.

[0022] The third step in the present invention is a step of dissolving the trifluoromethanesulfonate ester obtained in the second step in a third solvent and reacting it with a boron compound represented by general formula (4) or a boron compound represented by general formula (5) in the presence of a catalyst to produce an arylboronic acid ester represented by general formula (6).

[0023] [ka] (In the formula, R1 and R2 are the same or different and represent alkyl, aryl, alkoxy, or aryloxy having 1 to 6 carbon atoms, or R1 and R2 join together to form a cyclic catechol, pinacol, or bicyclononane; and R4, R5, R6, and R7 are the same or different and represent alkyl, alkoxy, or aryloxy having 1 to 6 carbon atoms.)

[0024] Specifically, a third solvent is added to the trifluoromethanesulfonate ester represented by the general formula (3), the boron compound represented by the general formula (4) or the boron compound represented by the general formula (5), and a palladium catalyst, and the mixture is heated under reflux. After the production of the product, the arylboronic acid ester represented by the general formula (6), is confirmed, the mixture is allowed to cool to room temperature, and a solvent such as ethyl acetate or distilled water is added to extract the product.

[0025] In this step, examples of the boron compound represented by the general formula (4) include bispinacolatodiboron and tetraalkoxydiboron, and examples of the compound represented by the general formula (5) include piconalborane.

[0026] In this step, a palladium catalyst is used. Examples of the palladium catalyst include PdCl2(dppe), PdCl2(dppp), and PdCl2(dppf). · Examples of suitable palladium catalysts include CH2Cl2, PdCl2(PPh3)2, PdCl2(o-tolyl)2, and Pd(PPh3)4. These palladium catalysts can be used alone or in combination. Examples of suitable cocatalysts include potassium acetate, ammonium acetate, diisopropylamine, trimethylamine, triethylamine, and tributylamine. These cocatalysts can be used alone or in combination.

[0027] The third solvent used in this step may be acetonitrile, 1,4-dioxane, or dimethyl sulfoxide, the heating reflux temperature is 60°C to 120°C, preferably 80°C to 100°C, and the reaction time is 6 hours to 24 hours, preferably 10 to 14 hours.

[0028] The fourth step in the present invention: a step of synthesizing Lp-boronophenylalanine by heating the arylboronic acid ester obtained in the third step in a fourth solvent under acidic conditions refers to a step of removing the protecting groups, diethylboron group and pinacol group, from the arylboronic acid ester to produce Lp-boronophenylalanine, as shown below.

[0029] [ka] (In the formula, R1 and R2 are the same or different and represent alkyl, aryl, alkoxy, or aryloxy having 1 to 6 carbon atoms, or R1 and R2 join together to form a cyclic catechol, pinacol, or bicyclononane; and R4, R5, R6, and R7 are the same or different and represent alkyl, alkoxy, or aryloxy having 1 to 6 carbon atoms.)

[0030] Specifically, the arylboronic acid ester represented by general formula (6) is heated under acidic conditions by adding hydrochloric acid or the like to a fourth solvent, if necessary, under an argon atmosphere at a heating temperature of 30°C to 70°C, preferably 40°C to 60°C, for 5 to 20 hours, preferably 10 to 14 hours.

[0031] The mixture obtained by the reaction is subjected to a conventional method such as ion exchange resin or high performance liquid chromatography (HPLC) to isolate and purify Lp-boronophenylalanine.

[0032] The enhancer used in tumor radiation therapy, which is characterized by containing Lp-boronophenylalanine produced by the above-mentioned manufacturing process as an active ingredient in the present invention, refers to a pharmaceutical composition containing Lp-boronophenylalanine, which is used as an enhancer in neutron capture therapy, as an active ingredient.

[0033] The pharmaceutical composition containing Lp-boronophenylalanine as an active ingredient may be a solid composition that dissolves when needed or a liquid composition that is diluted when needed, but a liquid composition is preferred.

[0034] The liquid composition may contain the active ingredient Lp-boronophenylalanine and a pharmaceutical base used in injections. It is also preferable to use an ionic liquid useful for dissolution, or fructose, sorbitol, or meglumine as a solubilizing agent.

[0035] The concentration of Lp-boronophenylalanine in the liquid pharmaceutical composition used as a complex is 1 to 30%, preferably 2 to 20%, more preferably 2 to 15%, and particularly preferably 2 to 10%, as a w / v concentration in the liquid composition. The boron concentration, converted to ppm, is 400 ppm to 15,000 ppm, preferably 900 ppm to 10,000 ppm, more preferably 900 ppm to 7,500 ppm, and particularly preferably 900 ppm to 5,000 ppm.

[0036] When an ionic liquid having high solubility is used, the concentration of Lp-boronophenylalanine in the liquid pharmaceutical composition is 15 to 50%, preferably 20 to 50%, more preferably 30 to 50%, and particularly preferably 30 to 45% w / v in the liquid composition. The boron concentration, converted to ppm, is 7200 ppm to 24000 ppm, preferably 9600 ppm to 24000 ppm, more preferably 14000 ppm to 21000 ppm, and particularly preferably 16000 ppm to 21000 ppm.

[0037] When meglumine is used as a solubilizing agent in the present invention, the molar ratio to Lp-boronophenylalanine is 1 or less, preferably 1 to 0.7, and particularly preferably less than 0.8 and 0.7 or more.

[0038] The pH of the liquid composition of the present invention is preferably near neutral, taking into consideration administration to a living body. More specifically, it is in the range of 6.5 to 7.5, and particularly preferably near 7.4. To adjust the pH, an appropriate pH adjuster (e.g., hydrochloric acid, sodium bicarbonate) or buffer used in the art may be used, as needed.

[0039] The osmotic pressure ratio of the liquid composition of the present invention is not particularly limited, but is preferably within the range of 1 to 2 relative to physiological saline, and more preferably within the range of 1.1 to 1.4. When the osmotic pressure ratio is within this range, in the case of an injection, it is possible to reduce pain and shorten the administration time.

[0040] The liquid composition may contain various metal ions, such as sodium and magnesium, contained in living bodies, as needed to enhance its stability in and outside the body. In the case of sodium ions, the concentration thereof is preferably in a range close to the range of sodium ion concentrations in body fluids so as not to significantly disrupt the electrolyte balance between intracellular fluid and extracellular fluid.

[0041] If necessary, the liquid composition may contain a buffer such as phosphate buffer, Tris-HCl buffer, acetate buffer, carbonate buffer, citrate buffer, etc. These buffers may be useful in stabilizing the formulation or reducing irritation.

[0042] Additives permitted as additives for liquid compositions under the Pharmaceutical and Medical Device Act can be contained as needed. Examples of such additives include additives typically used in liquid, particularly aqueous, compositions, such as preservatives such as benzalkonium chloride, potassium sorbate, and chlorhexidine hydrochloride, stabilizers such as sodium edetate, thickeners such as hydroxyethyl cellulose and hydroxypropyl methylcellulose, isotonicity agents such as sodium chloride, potassium chloride, glycerin, sucrose, and glucose, surfactants such as polysorbate 80 and polyoxyethylene hydrogenated castor oil, isotonicity agents such as sodium chloride, potassium chloride, and glycerin, and pH adjusters such as hydrochloric acid and sodium hydroxide.

[0043] When the liquid composition is a pharmaceutical used in neutron capture therapy, the liquid pharmaceutical composition, which contains Lp-boronophenylalanine as an active ingredient and an ionic liquid, may be administered by any method that delivers Lp-boronophenylalanine to the vicinity of the tumor, but preferably by intravenous administration, intraperitoneal administration, or transdermal administration.

[0044] In the case of administration methods involving direct injection into the body, such as intravenous administration or intraperitoneal administration, a dispersing agent such as polysorbate 80, polyoxyethylene hydrogenated castor oil 60, polyethylene glycol, carboxymethyl cellulose, or sodium alginate, a preservative such as methylparaben, propylparaben, benzyl alcohol, chlorobutanol, or phenol, or an isotonicity agent such as sodium chloride, glycerin, D-mannitol, or glucose may be added to a liquid pharmaceutical composition characterized by containing an ionic liquid consisting of p-boronophenylalanine, as needed, and the composition is diluted with a water-soluble solvent such as distilled water for injection, physiological saline, or Ringer's solution to produce a liquid pharmaceutical composition for intravenous or intraperitoneal administration, which contains p-boronophenylalanine as an active ingredient and is characterized by containing an ionic liquid.

[0045] Furthermore, when Lp-boronophenylalanine is to be delivered to the vicinity of a tumor by transdermal administration, a dispersing agent such as polysorbate 80, polyoxyethylene hydrogenated castor oil 60, polyethylene glycol, carboxymethylcellulose, sodium alginate, etc., a preservative such as methylparaben, propylparaben, benzyl alcohol, chlorobutanol, phenol, etc., or an isotonic agent such as sodium chloride, glycerin, D-mannitol, glucose, etc. may be added as necessary, and the resulting mixture may be dissolved, suspended, or emulsified in a vegetable oil such as olive oil, sesame oil, cottonseed oil, corn oil, etc., or an oily solvent such as propylene glycol, to produce a liquid pharmaceutical composition containing Lp-boronophenylalanine for transdermal administration as an active ingredient and an ionic liquid.

[0046] In the production process of the above pharmaceutical composition, additives such as solubilizing agents such as sodium salicylate and sodium acetate, stabilizers such as human serum albumin, and soothing agents such as benzyl alcohol may be used as desired, and further, antioxidants, colorants, solubilizing agents, suspending agents, isotonicity agents, buffers, soothing agents, preservatives, and gelling agents may be used as needed, provided that they are approved by the Pharmaceutical and Medical Device Act.

[0047] Furthermore, a liquid pharmaceutical composition containing Lp-boronophenylalanine as an active ingredient and an ionic liquid may be diluted with a water-soluble solvent such as distilled water for injection, physiological saline, or Ringer's solution, or with a pharmaceutical lotion or cream, if necessary, at the time of treatment.

[0048] A liquid pharmaceutical composition containing an ionic liquid and containing Lp-boronophenylalanine obtained by the production method of the present invention as an active ingredient can be produced by the following production method.

[0049] 1. A method for producing a liquid pharmaceutical composition containing Lp-boronophenylalanine as an active ingredient, meglumine, and an ionic liquid. At room temperature, meglumine is dissolved in pure water at a molar ratio of 2 or less, preferably 1 or less, and particularly preferably 0.2 to less than 0.8 relative to Lp-boronophenylalanine to a concentration of 0.002 mol / L to 0.02 mol / L. To this, p-boronophenylalanine is added in the amount specified relative to meglumine as described above, followed by further dissolution in pure water. The final concentration of p-boronophenylalanine is adjusted to 15 to 50 v / w%, preferably 20 to 50 v / w%, more preferably 30 to 50 v / w%, and particularly preferably 35 to 45 v / w%. If necessary, the aforementioned pharmaceutical formulation additives are added to produce a liquid pharmaceutical composition characterized by containing Lp-boronophenylalanine as an active ingredient and an ionic liquid. The pure water to be added may be commercially available pure water such as MiLLi-Q Water (trade name: manufactured by Merck Millipore).

[0050] 2. Formulation examples of liquid pharmaceutical compositions containing Lp-boronophenylalanine as a constituent Prescription example 1 To 65.45 mL of an ionic liquid composed of lp-boronophenylalanine and meglumine, 16.36 mL of Tween 80 (surfactant, Fujifilm Wako Pure Chemical Industries, Ltd.) and 65.45 mL of ethanol were added, and an appropriate amount of hydrochloric acid was added as a pH adjuster, followed by the addition of phosphate saline buffer (pH 7.4) to adjust the total volume to 654 mL.

[0051] Prescription example 2 To 100 μL of the ionic liquid composed of Lp-boronophenylalanine and meglumine, 10 μL of hydrochloric acid was added as a pH adjuster, and 890 μL of phosphate saline buffer (pH 7.4) was added to adjust the total volume to 1 mL, thereby producing a liquid pharmaceutical composition containing the ionic liquid composed of Lp-boronophenylalanine and meglumine. [Example]

[0052] Example 1 Preparation of Lp-boronophenylalanine First step [ka] L-tyrosine (451.6 mg, 2.50 mmol: Fujifilm Wako Pure Chemical Industries, Ltd.) was weighed into a flask. After adding a magnetic stirrer and tetrahydrofuran (17.5 mL: Kanto Chemical Co., Ltd.) to the flask, the atmosphere in the flask was replaced with argon, and triethylborane solution (ca. 1 mol / L in tetrahydrofuran, 7.5 mL, 7.50 mmol (3.01 eq): Kanto Chemical Co., Ltd.) was added while flowing argon. The flask was placed in an oil bath at approximately 100°C and heated to reflux for 41 hours, and the reaction solution was allowed to cool to room temperature. The reaction solution was concentrated under reduced pressure to obtain a colorless liquid. Hexane (approximately 50 mL) was added to the syrup-like crude product, and the resulting solid was suction filtered and washed with hexane. [ka] The boron ester represented by the formula (576.1 mg, 2.31 mmol, 92%) was obtained.

[0053] The resulting boron ester was identified as a single substance by TLC, and its structure was 1 H NMR and 13 This was confirmed by C NMR.

[0054] TLC readings Rf: 0.20 [hexane:ethyl acetate (1:1)] NMR analysis values 1 H NMR (500 MHz, DMSO-d6) δ: 0.10-0.23 (m, 4H), 0.62 (t, J = 7.8 Hz, 3H), 0.66 (t, J = 7.8 Hz, 3H), 2.81 (dd, J = 9.0, 14.5 Hz, 1H), 3.40 (dd, J = 4.0, 14.5 Hz, 1H), 3.69 (dddd, J = 4.0, 9.0, 9.0, 9.0 Hz, 1H), 5.23 (dd, J = 9.0, 11.2 Hz, 1H), 6.49 (dd, J = 6.4, 11.2 Hz, 1H), 6.70 (d, J = 9.0 Hz, 2H), 7.12 (d, J = 9.0 Hz, 2H), 9.24 (s, 1H). 13 C NMR (125 MHz, DMSO-d6) δ: 8.87, 9.06, 11.92, 12.55, 35.18, 56.12, 115.35, 127.20, 130.37, 156.25, 173.73

[0055] Second process The boron ester (3.4 g, 13.53 mmol) obtained in the first step and a magnetic stirrer were placed in a flask. The atmosphere inside the flask was replaced with argon, and then anhydrous acetonitrile (135 mL; Sigma-Aldrich) was added to the flask. Triethylamine (4.3 mL, 30.85 mmol (2.28 eq; Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the flask, and the flask was cooled to -40 °C. Trifluoromethanesulfonic anhydride (2.4 mL, 14.27 mmol (1.06 eq; Sigma-Aldrich) was added dropwise, and the reaction was allowed to proceed at the same temperature for 30 minutes. Saturated aqueous sodium bicarbonate (approximately 20 mL) was added, and the reaction mixture was allowed to warm to room temperature. Ethyl acetate (approximately 50 mL) was added, and the contents were transferred to a separatory funnel, and the ethyl acetate solution was separated. The resulting ethyl acetate solution was washed with 10% aqueous phosphoric acid, followed by saturated aqueous sodium bicarbonate, and then dried over anhydrous sodium sulfate. The drying agent was removed by gravity filtration, and the solvent was distilled off under reduced pressure using an evaporator to obtain a pale yellow oily substance. This crude product was dissolved in tetrahydrofuran (approximately 20 mL), and hexane (approximately 200 mL) was added to generate a white precipitate. This precipitate was collected by suction filtration, washed with hexane, and dried under reduced pressure to obtain a white oily substance. [ka] The trifluoromethanesulfonic acid ester represented by the formula: was obtained as white cotton-like crystals or colorless needles (3.7 g, 9.68 mmol, 72%).

[0056] The obtained trifluoromethanesulfonate ester was identified as a single substance by TLC, and its structure was 1 H NMR and 13 This was confirmed by C NMR.

[0057] TLC readings Rf: 0.34 (hexane:ethyl acetate (1:1)) NMR analysis values 1H NMR (500 MHz, DMSO-d6) δ: 0.11-0.30 (m, 4H), 0.670 (t, J = 7.5 Hz, 3H), 0.674 (t, J = 7.8 Hz, 3H), 2.93 (dd, J = 10.0, 15.0 Hz, 1H), 3.24 (dd, J = 4.0, 15.0 Hz, 1H), 3.86 (m, 1H), 5.61 (dd, J = 9.5, 11.0 Hz, 1H), 6.56 (dd, J = 7.5, 11.0 Hz, 1H), 7.45 (d, J = 8.8 Hz, 2H), 7.56 (d, J = 8.8 Hz, 2H). 13 C NMR (125 MHz, DMSO-d6) δ: 8.84, 8.97, 11.64, 12.45, 35.32, 55.37, 118.26 (q, JC-F = 68.8 Hz), 121.21, 131.55, 138.68, 148.09, 173.23.

[0058] Third Project The trifluoromethanesulfonate ester (380.0 mg, 1.00 mmol) obtained in the second step, PdCl(dppf).CHCl (41.0 mg, 0.05 mmol (5 mmol%): Sigma-Aldrich), bis-pinacolatodiboron (383.2 mg, 1.51 mmol (1.51 eq): Sigma-Aldrich), and potassium acetate (395.7 mg, 4.00 mmol (4.04 eq): Kanto Chemical Co.) were weighed into a flask. The atmosphere in the flask was replaced with argon, and anhydrous acetonitrile (3.3 mL: Sigma-Aldrich) was added. The flask was placed in an oil bath preheated to 100 °C and refluxed for 12 hours. The reaction mixture was then allowed to cool to room temperature. The reaction mixture was diluted with approximately 10 mL of ethyl acetate, transferred to a separatory funnel, and distilled water was added. The ethyl acetate solution was then separated. The ethyl acetate solution was transferred to a 50 mL Erlenmeyer flask and dried over anhydrous sodium sulfate, after which the desiccant was removed by gravity filtration. The solvent was removed under reduced pressure using an evaporator to obtain a black oily substance. This crude product was dissolved in a small amount (approximately 10 mL) of ethyl acetate, and after adding an approximately equal amount of hexane, the solution was passed through a silica gel pad (15 g of silica gel). [ka] An arylboronic acid ester (351.1 mg, 0.98 mmol, 98%) having the structure shown in the following was obtained (silica gel was eluted with a mixed solution of hexane and ethyl acetate (2:1), and the fractions containing the product were confirmed by TLC, collected, and concentrated).

[0059] The resulting arylboronic acid ester was identified as a single substance by TLC, and its structure was 1 H NMR and 13 This was confirmed by C NMR.

[0060] TLC readings Rf: 0.60 [hexane:ethyl acetate (1:1)] NMR analysis values 1H NMR (500 MHz, DMSO-d6) δ: 0.11-0.26 (m, 4H), 0.647 (t, J = 7.8 Hz, 3H), 0.653 (t, J = 7.8 Hz, 3H), 2.91 (dd, J = 9.3, 14.6 Hz, 1H), 3.19 (dd, J = 4.0, 14.6 Hz, 1H), 3.77 (dddd, J = 4.0, 8.5, 8.5, 8.5 Hz, 1H), 5.52 (dd, J = 9.5, 11.2 Hz, 1H), 6.50 (dd, J = 7.8, 11.2 Hz, 1H), 7.36 (d, J = 8.0 Hz, 2H), 7.62 (d, J = 8.0 Hz, 2H). 13 C NMR (125 MHz, DMSO-d6) δ: 9.00, 9.10, 11.72, 12.57, 24.67, 36.28, 55.78, 83.77, 126.71 (br), 128.94, 134.73, 141.15, 173.62

[0061] Fourth step The arylboronic acid ester (413.8 mg, 1.15 mmol) obtained in the third step was weighed into a flask, and 1,4-dioxane (2.3 mL: manufactured by Kanto Chemical Co., Ltd.) and 2 mol / L hydrochloric acid (3.0 mL, 6.00 mmol (5.21 eq): manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were added to the flask. After the atmosphere in the flask was replaced with argon, the flask was immersed in an oil bath preheated to 50°C and heated for 14 hours. After cooling to room temperature, the reaction mixture was passed through a cation exchange resin (Dowex 50, H+ type, resin volume 50 mL). After washing the resin with distilled water, lp-boronophenylalanine was eluted with 2% aqueous ammonia solution. The fractions containing lp-boronophenylalanine were collected, concentrated under reduced pressure using an evaporator, and dried under reduced pressure to obtain the desired product. [ka] Lp-boronophenylalanine (205.8 mg, 0.98 mmol, 98%) shown below was obtained.

[0062] The structure of the obtained Lp-boronophenylalanine is: 1 H NMR and 13 This was confirmed by C NMR.

[0063] NMR analysis values 1 H NMR (500 MHz, D2O + DCl) δ: 3.02 (dd, J = 7.5, 14.4 Hz, 1H), 3.13 (dd, J = 5.8, 14.4 Hz, 1H), 4.16 (dd, J = 5.8, 7.5 Hz, 1H), 7.11 (d, J = 8.0 Hz, 2H), 7.50 (d, J = 8.0 Hz, 2H). 13 C NMR (125 MHz, D2O + DCl) δ: 35.75, 54.08, 129.23, 131.86 (br), 134.60, 136.86, 171.26

[0064] The yield of Lp-boronophenylalanine from L-tyrosine was as high as 63.6%.

[0065] Example 2 Neutron capture therapy enhancing effect of Lp-boronophenylalanine obtained in this invention 187.7 mg of fructose and 600 μL of 1 mol / L aqueous sodium hydroxide solution were added to 84.4 mg of Lp-boronophenylalanine obtained in Example 1 and dissolved, and then the pH was adjusted to 7.2 with 1 mol / L hydrochloric acid. The boron concentration of the resulting solution was adjusted to 1000 ppm by adding distilled water, and this was used as the test compound (hereinafter referred to as L-nBPA).

[0066] BALB / cA mice (female, 4 weeks old, weighing 16-20g) were used as tumor-bearing mice, and mouse colon cancer cells (CT26.5x10 6 After transplantation into the right thigh, the mice were raised for 12 days, and tumors were formed with a diameter of 6 to 8 mm (average tumor volume 230 mm). 3The tumor-bearing mice were divided into three groups (n = 4-6 per group): an untreated group that received no treatment during the test, an irradiated group (control group) that received only neutron beam irradiation during the test, and an L-nBPA-administered group that received neutron beam irradiation after administration of L-nBPA. The test compounds were administered to the tumor-bearing mice as follows, and the therapeutic enhancement effect and antitumor effect during boron neutron capture therapy (BNCT) were examined.

[0067] The test compound was administered via the tail vein at a concentration of 10 mgB / kg in terms of boron. No pre-administration was performed in the control group irradiated with neutrons alone and in the untreated group.

[0068] Two hours after administration of the test compound, neutron irradiation was carried out at the Kyoto University Research Reactor (KURNS). The neutron dose was 1.3-3.6×10 12 neutrons / cm 2 The irradiation time was 50 minutes. The tumor suppression effect was evaluated by measuring the tumor diameter over time up to the 28th day after irradiation and comparing it with the control group. The results are shown in Table 1 and Figure 1.

[0069] [Table 1]

[0070] In the above test, the tumor size was measured according to the following calculation formula. [Long diameter (mm)] x [Short diameter (mm)] 2 / 2 = tumor size (mm 3 ) As a result, the L-nBPA group was especially suitable for neutron capture therapy. 10 Although B was not highly concentrated, it significantly suppressed tumor growth compared to the control group. [Industrial Applicability]

[0071] INDUSTRIAL APPLICABILITY The present invention provides Lp-boronophenylalanine that can be used in neutron capture therapy without high concentration.

Claims

1. A method for producing L-p-boronophenylalanine, comprising the following four steps: First step: a step of reacting L-tyrosine with a boron derivative represented by general formula (1), such as triethylborane or tributylborane, in an ether solvent such as tetrahydrofuran or 1,4-dioxane at a temperature equal to or higher than the boiling point for 12 to 96 hours to produce a boron ester represented by general formula (2); 【Chemistry 1】 (wherein R 1 , R 2 and R 3 are the same and represent alkyl having 2 or 3 carbon atoms.) Second step: dissolving the boron ester obtained in the first step in acetonitrile, 1,4-dioxane or cyclopentyl methyl ether, cooling the solution to −20° C. to −60° C. in the presence of a trialkylamine, and then reacting the solution with trifluoromethanesulfonic anhydride for 15 to 60 minutes to produce a trifluoromethanesulfonic acid ester represented by general formula (3); 【Chemistry 2】 (wherein R 1 , R 2 and R 3 are the same and represent alkyl having 2 or 3 carbon atoms.) Third step: The trifluoromethanesulfonate ester obtained in the second step is dissolved in acetonitrile, 1,4-dioxane or dimethyl sulfoxide, and reacted with a boron compound represented by general formula (4) or a boron compound represented by general formula (5) in the presence of a palladium catalyst selected from PdCl 2 (dppe), PdCl 2 (dppp), PdCl 2 (dppf).CH 2 Cl 2 , PdCl 2 (PPh 3 ) 2 and Pd(PPh 3 ) 4 at 60° C. to 120° C. for 6 to 24 hours to produce an arylboronic acid ester represented by general formula (6). 【Transformation 3】 (wherein R 1 and R 2 are the same and represent alkyl having 2 to 3 carbon atoms, and R 4 , R 5 , R 6 and R 7 are the same and represent alkyl having 1 to 2 carbon atoms.) and Fourth step: A step of producing L-p-boronophenylalanine by reacting the arylboronic acid ester obtained in the third step in dioxane under acidic conditions at 30°C to 70°C for 5 to 20 hours. 【Chemistry 4】 (wherein R 1 and R 2 are the same and represent alkyl having 2 to 3 carbon atoms, and R 4 , R 5 , R 6 and R 7 are the same and represent alkyl having 1 to 2 carbon atoms.)

2. A method for producing L-p-boronophenylalanine according to claim 1, characterized in that the boron compound is bis-pinacolatodiboron.

3. The method for producing L-p-boronophenylalanine according to claim 1, characterized in that the boron compound is pinacolborane.

4. The method for producing L-p-boronophenylalanine according to any one of claims 1 to 3, wherein the trialkylamine used in the second step is trimethylamine, triethylamine, tributylamine, or diisopropylethylamine.

5. The method for producing L-p-boronophenylalanine according to any one of claims 1 to 3, wherein the catalyst used in the third step is a palladium catalyst, and the co-catalyst is potassium acetate, ammonium acetate, diisopropylamine, or one or a combination of potassium acetate, ammonium acetate, diisopropylamine, trimethylamine, triethylamine, and tributylamine.

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